<p class="jove_content">Geçerli proje kavramı Fonu University of Kansas kanıtı tarafından finanse edildi. Biz bize cochleae kültürleri ile yardım için insan göbek bağları ve David Jorgensen elde yardımcı olmak için KUMC (Kansas City, KS) hemşirelerin teşekkür etmek istiyorum.</p>

<p class="jove_content">Tüm yordamları, hayvan ötenazi, dahil olmak üzere onaylanmış kurumsal hayvan bakım ve kullanım Komitesi (IACUC) iletişim kuralı (ACUP #2014-2234) Kansas Üniversitesi Tıp Merkezi (KUMC) göre yapılmıştır.</p>
<p class="jove_content">Not: HWJCs Onam sağlanan hastalar tarafından bağışlandı insan göbek bağları yalıtılmış ve numuneler Kansas Üniversitesi insan denekler Komitesi tarafından (KU-IRB #15402) onaylanmış protokolleri uygun olarak kullanılmıştır.</p>
<p class="jove_title">1. Temporal kemik hasat ve koklea yalıtım</p>
<ol>
	<li>Cerrahi makas keskin bir çift ile kafatası ve ilk servikal omur arasında keserek uygun şekilde ötenazi, 15-hafta-yaşlı fare başını kesmek, o zaman (<strong class="xfig">şekil 1A</strong>) dezenfekte için % 70 etanol ile baş aşağı sprey.</li>
	<li>Cerrahi makas (<strong class="xfig">şekil 1B</strong><strong>-C</strong>, kesik çizgiler) aynı keskin çiftini kullanarak bir-sagittal düzlemde kafatası bisect.</li>
	<li>Beyin dokusu forseps (<strong class="xfig">şekil 1 d</strong>, ok ucu) bir çift kullanarak kafatasından kaldırın.</li>
	<li>Temporal kemik işitsel varlığı ve kafatası ve (<strong class="xfig">şekil 1F</strong>, ok) dış kulak kanalına iç vestibular sinir kökleri (<strong class="xfig">şekil 1E</strong><strong>-F</strong>, ok uçları) tanımlayın. Dikkatle kafatası kafatası (<strong class="xfig">şekil 1G</strong>) kalan şakak kemiğinden yalıtmak için kesti.<br>Not: bazı durumlarda, ince kesim gerek kalmadan kafatası temporal kemik uzak çevresindeki kemiklerin burnumu sokmak mümkün olabilir.</li>
	<li>Böylece ipuçları koklea delinme riski değil iyi forseps kulak kanalına yaklaşık 5 mm (<strong class="xfig">şekil 1 H</strong>), açılış içine ekle. Yavaşça bulla (<strong class="xfig">şekil 1 H</strong>, en iyi kırmızı gölgeli alan) kemik kır aç (noktalı<strong class="xfig">1I rakam</strong>, kırmızı hat) kırıkları bu yüzden.<br>Not: gerekirse, diseksiyon mikroskop bu süreçte doğru yerleştirme ve işleme aletleri sağlamak için yardımcı olabilir.</li>
	<li>İyi forseps kullanarak, uzak bulla temporal kemikten, koklea (<strong class="xfig">şekil 1J</strong>, kırmızı köşeli ayraçlar) kemik labirent maruz kalan gözetlemek.</li>
	<li>Temporal kemik alacak kadar büyük bir Petri kabına yerleştirin (<em>örneğin</em>, 30 mm veya daha büyük) altında diseksiyon kapsam görüş alanı ve yeterli fosfat tamponlu tuz (temporal kemik (<em>örneğin</em>, 0.5-1 mL) kapsayacak şekilde PBS ile) doldurun.</li>
	<li>Yukarı dönük olacak şekilde koklea oval ve yuvarlak pencerelerle PBS kaldırıldı bulla ile temporal kemik daldırın.<br>Not: Vestibüler sistem manipüle ve koklea (<strong class="xfig">1I anlamaya</strong> ve <strong class="xfig">şekil 2B</strong>) özelliklerini yönlendirme için de bir kolu olarak hizmet vermektedir olarak koklea Vestibüler sistemden kaldırılması gerekli değildir.</li>
	<li>Forseps Ultra-ince bir çifti kullanarak, stapes geçer stapedial arter kaldırın.</li>
	<li>Ultra-ince forseps nerede arter geçtiğini üzengi kemer aracılığıyla bir ucu yerleştirin ve ince üzengi yukarı doğru kaldırın. Disarticulated üzengi oval pencere kapalı kaldırmak.</li>
	<li>Ultra-ince forseps bir ucu kullanarak, oval ponksiyon ve windows yuvarlak.</li>
	<li>1 x PBS kullanarak 28.5'lik şırınga ile tüp dolu (iç çap 0,28 mm, dış çap 0,61 mm) bağlı, tüp oval penceresinin üzerine getirin (<strong class="xfig">şekil 2A</strong> <strong> -B</strong>).<br>Not: Tüp açılış yardımcı olmak doğru yerleşim sağlamak için ultra-ince forseps kullanarak manipüle edilebilir.<ol>
		<li>Temiz şırınga ve boru her koklea için kullanın.</li>
	</ol>
	</li>
	<li>% 10 antibiyotik antimycotic (anti-anti) ve % 10 penisilin-streptomisin (kalem-strep) PBS içinde koklea 5 dk ile perilymph kaldırmak için 2 mL sıvı. Çok hızlı çok fazla baskı ile Ventriküler koklea içinde güzel yapıları zarar verir.<br>Not: bir perfüzyon pompası alternatif olarak kullanılabilir, ancak el ile el ile koklea yoluyla sıvı sıvı şırınga sürüş etkilidir.<ol>
		<li>El ile Ventriküler, koklea (<strong class="xfig">şekil 2A</strong>) dengelemeye forseps perfüzyon sırasında kendi kendine kapanış temporal kemik Vestibüler bölümünü açgözlü bir çift iyi kullanın.<br>Not: gerekli değildir, ancak bu iki el aletleri işlemek serbest bırakır; bir yandan diğer tüp konumlandırabilirsiniz iken şırınga (<strong class="xfig">şekil 2A</strong>) sürebilirim uygun şekilde (<strong class="xfig">şekil 2B</strong>).</li>
		<li>Bu adım ileri, koklea gereksiz yere hava için kullanılmasını önlemek için dikkat ediniz. Hava kabarcıkları sıvı alanlarda tanıtımı sıvı dolaşımını engeller ve daha fazla kurumasını koklea içinde güzel yapıları zarar verir.</li>
	</ol>
	</li>
	<li>Kaldırmak ve iyi forseps kullanarak herhangi bir kalan kas doku ve kemik parçaları atın.</li>
	<li>Gerekirse, izole cochleae 4 ° C'de % 10 anti-anti ve % 10 kalem-strep çözümde PBS antibiyotik çözüm her 48 h normal değişikliklerle yedi güne kadar saklayın.</li>
</ol>
<p class="jove_title">2. koklear işleme</p>
<ol>
	<li><strong>Decellularization</strong>
	<ol>
		<li>Her koklea için dolgu 20 mL cam mercek şişe ile % 1 Sodyum Lauryl Sülfat (SDS) çözüm koklea decellularize eskiden de-iyonize (DI) su içinde.</li>
		<li>Kapalı açılış geçmesine bir koklea için yeterli büyüklükte bir jilet kullanarak bir plastik 7-mL transfer pipet ucu kesilmiş.</li>
		<li>Sadece bir kaç milimetre kesme kenarına geçer böylece transfer pipet kullanarak yavaşça koklea pipet çizin.</li>
		<li>Koklea % 1 SDS çözüm dolu mercek flakon kovmak.</li>
		<li>2 mL % 1 SDS koklea transfer pipet kullanarak mercek şişe içinde de-iyonize (DI) su içinde dolaşımda.</li>
		<li>Mercek şişe bir rotator yerleştirin ve oda sıcaklığında 72 h için 10 RPM döndürmek mercek şişe sağlar.<ol>
			<li>% 1 SDS çözüm her 24 h bir 72 saat süre içinde değiştirin. SDS koklea hava için pilleri değiştirirken dikkat et.</li>
		</ol>
		</li>
		<li>Koklea üç kez için 30 dk her DI su ile yıkayın.</li></ol></li></ol>SDS giderler için kullanılan aynı mercek şişe yıkama gerçekleştirmek; Bu aşamada, koklea decellularized.
	
	
	<li><strong>Kalsiyumun</strong>
	<ol>
		<li>Kalan DI su batık koklea tutmak yeterli bırakarak mercek şişeyi kaldırın.</li>
		<li>Kalsiyumun başlamak için koklea içeren mercek şişeyi DI suda 5 mL % 10 ethylenediaminetetraacetic asit (EDTA) (0,02 M) de ekleyin.</li>
		<li>2 mL % 10 EDTA DI suyun içinden bir transfer pipet kullanarak mercek şişe koklea içinde dolaşımda.</li>
		<li>Mercek şişeyi geri rotator içinde yerleştirin ve oda sıcaklığında 72 h için 10 RPM döndürmek mercek şişe sağlar. % 10 EDTA çözüm her 24 h bir 72 saat süre içinde değiştirin. Böylece koklea havaya maruz değil çözümleri değiştirirken dikkat et.</li>
		<li>1 x PBS ile her 2 h için 3 kez koklea durulayın; Bu aşamada, koklea decalcified.</li>
	</ol>
	</li>
	<li><strong>Depolama</strong>
	<ol>
		<li>Cochleae en fazla 72 saat için % 10 anti-anti, % 10 kalem-strep çözeltilerine PBS 4 ° C'de depolayın<br>Not: Daha uzun depolama süre antibiyotik çözüm düzenli değişiklikler ile mümkün olabilir; Ancak, genişletilmiş depolama işlenmiş cochleae, bu protokol için doğrulanmadı.</li>
	</ol>
	</li>

<p class="jove_title">3. satın alma ve hWJCs genişlemesi</p>
<ol>
	<li>HWJCs göre daha önce yayımlanmış protokolleri<sup class="xref">22</sup>yalıtmak. Kısaca, göbek bağları 3 cm bölümlere kesiminde.</li>
	<li>Dikkatli bir neşter ile sığ bir kesi boyuna göz önüne sermek ve göbek kordonu Wharton'ın jöle maruz her göbek kordonu kesiminde olun. Forseps kan damarları kaldırmak için kullanın.</li>
	<li>Göbek kordonu kesimleri yeterince PBS (<em>örneğin</em>, 60 mm Petri kabına 40 mL) ile iki kez doku daldırın yıkayın. İnce doku parçaları bir neşter ile kıyma. Sindirmek doku 100 mm Petri dish ile sindirim orta 50 mL (% 0,2 tip 2 collagenase, % 1 penisilin-streptomisin, düşük glikoz içinde Dulbecco'nın modifiye kartal orta (DMEM)).</li>
	<li>Doku bir orbital Çalkalayıcı geceleme bir % 5 CO<sub>2</sub>, 37 ° C kuluçka 50 RPM medyada sindirerek yerleştirin. Ertesi gün, seyreltik sindirim orta 1:16 antibiyotik-antimycotic PBS %2 oranında ve cips hücreleri tarafından Santrifüjü vasıl 500 x g oda sıcaklığında (~ 27 ° C) için 10 dk. atma süpernatant.</li>
	<li>Granül Mezenkimal Kök hücre büyüme orta (MSCGM) resuspend ve hücreleri birleştireceğimi. 7 x 10<sup>3</sup> hücreleri/cm<sup>2 </sup>doku kültürü'yoğunluğu plaka hücreleri T-75 şişeler tedavi. HWJCs MSCGM içinde kültür ve geçit 5 deneyler için genişletin.<br>Not: HWJCs daha büyük T-şişeler alt kültürlü (<em>örneğin</em>, T-150, T-300) hWJCs deneyler için verimini artırmak için passaging zaman.</li>
</ol>
<p class="jove_title">4. Decellularized Cochleae içine hWJCs infüzyonu</p>
<ol>
	<li><strong>Decellularized cochleae hazırlamak</strong>
	<ol>
		<li>Aseptik tekniği kullanarak, saklı cochleae 1 x PBS 30 dk için üç kez yıkayın.</li>
		<li>Cochleae 24-şey plaka ayrı kuyu aktarmak ve 37 ° C 1 mL ekleyin önceden MSCGM ısıttı.</li>
		<li>Cochleae bir % 5 CO<sub>2</sub>, 37 ° C hücre kültür kuluçka en az 1 h önce hücreleri infüzyonu kuluçkaya.</li>
	</ol>
	</li>
	<li><strong>HWJCs ayırmak ve resuspend</strong>
	<ol>
		<li>Yıkama hWJCs 37 ° C ile 1 x PBS iki kez önceden ısıttı.</li>
		<li>Yeterli 37 ° C eklemek önceden tripsin hücre gemi yüzeyi kapsayacak şekilde % 0.5 EDTA ile ısıttı.</li>
		<li>HWJCs 5 dakikaya kadar bir % 5 CO<sub>2</sub>, 37 ° C hücre kültür kuluçka için kuluçkaya.<ol>
			<li>Ters bir mikroskop altında hücreler görüntüleyerek %90 hücre hücre kültür yüzeyden ayırdıktan doğrulayın.<br>Not: Kültür gemi hafifçe vurdu olduğu zaman, hareket hücreleri ilişkisi kesildi. Sabit kalır, değil ayırdıktan hücreler.</li>
			<li>Daha fazla bağlı hücreler çıkarmak için balonun kenarlarına hafifçe dokunun.</li>
		</ol>
		</li>
		<li>Aseptik teknik kullanılarak, kültür gemisinden transfer hWJCs içeren MSCGM eşdeğer bir birimi tripsin hacmiyle 50 mL konik tüp için hücreleri ayırmak için kullanılır.</li>
		<li>500 x g oda sıcaklığında, centrifuging tarafından hWJCs cips (~ 27 ° C) 5 min için.</li>
		<li>Süpernatant Aspire edin ve hWJCs bir konsantrasyon 500.000 hücre/mL MSCGM resuspend.</li>
	</ol>
	</li>
	<li><strong>Cochleae sıvı</strong>
	<ol>
		<li>Cochleae transfer pipet kullanarak yeni bir 24-şey tabağa aktarın.</li>
		<li>MSCGM bir damla herhangi bir koklea kurumasını önlemek için her koklea ekleyin.</li>
		<li>İnce oval ve yuvarlak windows karşı karşıya yükleyebilmeniz için ultra-ince forseps kullanarak koklea gelecek şekilde yönlendirin.<br>Not: koklea kolayca zarar gibi her koklea doğrudan işlerken aşırı kendine iyi bak.</li>
		<li>Steril 28.5'lik insülin ile bağlı tüp kullanarak, 0.2 mL resuspended hWJCs (100.000 hücreleri) kadar çizin.</li>
		<li>Steril iyi forseps kullanarak, boru oval penceresinin üzerine getirin.</li>
		<li>İnce ve yavaş yavaş koklea ile resuspended hWJCs polietilen boru için bağlı 28.5'lik insülin kullanarak 0.2 mL sıvı (dış çap: 0,61 mm, iç çapı: 0,28 mm) yaklaşık 5 dk içinde.</li>
		<li>Perfüzyon sonra 37 ° C 0.8 mL ekleyin önceden sıcak MSCGM de 1 mL su kuyusunda Toplam hacim getirmek koklea içeren için.</li>
		<li>Adımları 4.3.3-4.3.7 taze bir Ģırınga kullanarak ve her zaman boru her koklea için yineleyin.</li>
		<li>Yer cochleae %5 CO<sub>2</sub>, 37 ° C hücre kültür kuluçka ve değişim medya haftada üç kez derin.</li>
	</ol>
	</li>
</ol>
<p class="jove_title">5. koklea hasat ve koruma</p>
<p class="jove_content">Not: Cochleae kültürlü olabilir ve herhangi bir zaman noktasında 30 gün sonrası perfüzyon hasat.</p>
<ol>
	<li>Cochleae korumak için 1 x PBS gecede 4 ° C'de sallanan bir platformda %4 paraformaldehyde düzeltin.</li>
	<li>Fiksasyon sonra cochleae 1 x PBS 5 min için üç kez ile yıkayın.</li>
	<li>Yavaş yavaş koklea etanol ile kurutmak ve parafin içinde katıştırma önce bir Alifatik hidrokarbon çözücü ile temizleyin.</li>
	<li>Örnekleri için bir microtome kullanarak 10 µm kalınlığında bölüm ve cam mikroskop slaytlar üzerine monte edin.<br>Not: Örnekleri için hazırsınız histolojik veya immunohistokimyasal işleme standart protokolleri kullanarak.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+extracellular+matrix+in+the+formation+of+the+inner+ear.">Involvement of extracellular matrix in the formation of the inner ear.</a> <em style='font-style: italic'>Dev Dyn</em>. <b>202</b> (4), 421-432 (1995).</li><li>Goodyear, R. J., Richardson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrices+associated+with+the+apical+surfaces+of+sensory+epithelia+in+the+inner+ear:+molecular+and+structural+diversity.">Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structural diversity.</a> <em style='font-style: italic'>J Neurobiol</em>. <b>53</b> (2), 212-227 (2002).</li><li>Mellott, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+Viability+and+Transfection+Efficiency+with+Human+Umbilical+Cord+Wharton's+Jelly+Cells+Through+Use+of+a+ROCK+Inhibitor.">Improving Viability and Transfection Efficiency with Human Umbilical Cord Wharton's Jelly Cells Through Use of a ROCK Inhibitor.</a> <em style='font-style: italic'>Cell Reprogram</em>. , (2014).</li><li>Mellott, A. J., Shinogle, H. E., Moore, D. S., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Photo-conversion:+A+second+chance+to+label+unique+cells.">Fluorescent Photo-conversion: A second chance to label unique cells.</a> <em style='font-style: italic'>Cell Mol Bioeng</em>. <b>8</b> (1), 187-196 (2015).</li><li>Mitchell, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+cells+from+Wharton's+jelly+form+neurons+and+glia.">Matrix cells from Wharton's jelly form neurons and glia.</a> <em style='font-style: italic'>Stem Cells</em>. <b>21</b> (1), 50-60 (2003).</li><li>Mellott, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+Reprogramming+of+Human+Wharton's+Jelly+Cells+Reveals+Differences+Between+ATOH1+Homologues.">Nonviral Reprogramming of Human Wharton's Jelly Cells Reveals Differences Between ATOH1 Homologues.</a> <em style='font-style: italic'>Tissue Eng Part A</em>. <b>21</b> (11-12), 1795-1809 (2015).</li><li>Buytaert, J. A., Johnson, S. B., Dierick, M., Salih, W. H., Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+versus+sTSLIM+3D+imaging+of+the+mouse+cochlea.">MicroCT versus sTSLIM 3D imaging of the mouse cochlea.</a> <em style='font-style: italic'>J Histochem Cytochem</em>. <b>61</b> (5), 382-395 (2013).</li><li>Nonoyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+ototoxicity+of+gadoteridol+(ProHance)+and+gadodiamide+(Omniscan)+in+mice.">Investigation of the ototoxicity of gadoteridol (ProHance) and gadodiamide (Omniscan) in mice.</a> <em style='font-style: italic'>Acta Otolaryngol</em>. <b>136</b> (11), 1091-1096 (2016).</li><li>Dai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhesus+Cochlear+and+Vestibular+Functions+Are+Preserved+After+Inner+Ear+Injection+of+Saline+Volume+Sufficient+for+Gene+Therapy+Delivery.">Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery.</a> <em style='font-style: italic'>J Assoc Res Otolaryngol</em>. , (2017).</li><li>Sutherland, A. J., Converse, G. L., Hopkins, R. A., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioactivity+of+cartilage+extracellular+matrix+in+articular+cartilage+regeneration.">The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.</a> <em style='font-style: italic'>Adv Healthc Mater</em>. <b>4</b> (1), 29-39 (2015).</li></ol>

<p class="jove_content">Yazarlar ifşa gerek yok.</p>

<p class="jove_content">Yerel koklear hücreleri koklea koklea bir karmaşık, üç boyutlu doku iskele olarak kullanmak için izin verir bir decellularization süreci ile gelen kaldırılabilmesi için başarılı bir şekilde göstermiştir. Santi <em>ve ark</em>. <sup class="xref">15</sup> decellularizing cochleae için ilk yöntem geliştirdi ve birçok koklear yapıları birimleri hafif levha mikroskobu<sup class="xref">23</sup>yardımı ile doğru bir şekilde tahmin. Böyle erken dönem çalışmaları doku Mühendisliği ve burada sunulan hücre kültürü teknikleri için güçlü bir temel olarak görev yaptı. Decellularized koklea başarıyla hücrelerle infüzyon olabilir ve uzun bir süre için kültürlü. Cochleae hayvanlar geniş bir dizi hücre türleri benzer şekilde geniş bir dizi ile birlikte teorik olarak yararlanılabilir. Burada çok sayıda mümkün deneysel tasarımlar, doku Mühendisliği duyu epiteli geliştirme<sup class="xref">14</sup>incelemek, yeni ilaç ajanları<sup class="xref"> test ilaç uygulamaları gibi kullanılmak üzere sunulan teknikleri bu birleşimlerine izin vermek 24</sup>, ve geliştirme koklear onarım model<sup class="xref">25</sup>. Ancak, bu teknikler geniş uygulanabilirliği rağmen onlar sınırlamalar değildir.</p>
<p class="jove_content">Bir geçerli teknik sınırlamaları perfüzyon bireyin temel değişken olmasıdır. Bir sahibi, koklea için geliştirme ve perfusions yapmak için bir şırınga pompa kullanma doğruluk ve sürecinin başarısı artabilir. Ayrıca, başarılı decellularization kesin olarak ispat potansiyel olarak çeşitli yöntemlerle başarılı olabilir. Biz DAPI kalan hücre çekirdeği ölçmek için kullanılan boyama istihdam ve biz hiçbir kalıntı çekirdek yazı-decellularization gözlenen. Ayrıca, standart DNA miktar deneyleri kullanmak reaktif, yeşil boya picogram miktarda DNA nükleik asit kalıntıları, görsel olarak küçük parçaları nükleik varlığını belirlemek için DAPI daha daha duyarlı olabilir belirlemek için tespit decellularized doku<sup class="xref">26</sup>asitleri. Hangi teknikleri başarılı decellularization doğrulamak için kullanılan ne olursa olsun, bu bir iskele kullanan önce bireysel bir koklea tam decellularization göstermek mümkün olmayabilir. Yine de, biz decellularization işleminin başarılı ve sağlam olması için bulduk.</p>
<p class="jove_content">Geçerli protokol en kritik iki adımda yalıtım ve cochleae işlenmesi ve cochleae perfüzyon vardır. Koklea hassas ve kırılgan olduğu gibi koklea başlangıçta izole olduğunda büyük özen göstermelidir. Çok fazla kuvvet Forseps ile koklea işlerken uygulanmışsa, koklea parçası. Böylece, her koklea hafifçe decellularization ve incelemelerine daha önce işlemek için zorunludur. Vestibüler sistem, sol bozulmamış, eğer karmaşık iç kulak Forseps ile kapma ve koklea yönlendirme için büyük bir kolu olarak hizmet vermektedir. Benzer şekilde, koklea hücrelerle Ventriküler arkasında perfüzyon basıncı çok büyük olmaması gerekir, aksi takdirde hücreleri perfüzyon hayatta değil. Geçerli protokol belirtildiği gibi koklea ile bir yandan ve diğer elinizle koklea ile hücreleri Ventriküler daha yüksek hassasiyet elde etmek daha kolay işleme boru 1 mL insülin şırınga sonuna ekleme sağlar.</p>
<p class="jove_content">Bir sonraki adım olarak, koklear ECM kök hücre farklılaşma inducing ve çok, hangi belirli ECM bileşenleri koklea içinde hücrelerdeki değişiklikler inducing belirlemek için gelişimsel ve işlev işaretçileri değerlendirmek için doğal olurdu. Koklea monitör hücre farklılaşma ve davranış ile hücreleri (<em>örneğin</em>, kök hücreler, neuroprogenitors, fibroblastlar, <em>vb</em>) farklı türde Ventriküler başka bir büyüleyici soruşturma olur. Buna ek olarak, genetik olarak değiştirilmiş veya bir büyüme faktörü farklılaşma iletişim kuralına maruz hücreler nasıl koklear ECM neurosensory geliştirmesini destekler içine yeni anlayışlar sağlamak ve işitme işlevinde hatta. Böylece, geçerli protokol işitme kaybı geri yüklemek için yeni tedavilerin geliştirilmesi için bir gün kurşun iç kulak neurosensory geliştirme ve bakım, eğitim için koklear ECM kullanarak bir önemli ilk adımdır.</p>

<p class="jove_content">Koklea temporal kemik bulundu bir karmaşık sarmal yapısıdır. Bir dış kemik labirent ve konsantrik, iç membranöz labirentin<sup class="xref">1</sup>oluşur. Membranöz labirentin üç sıvı boşluk oluşur: Scala vestibuli, Scala medya ve Scala tympani<sup class="xref">1</sup>. Çok sayıda hücre tiplerinin oluşmaktadır, duyu epiteli scala medya evler ama duyusal saç ses dalgaları sinir uyarılarının<sup class="xref">2</sup>mekanik enerji transduce, hücreleri (HC), belirli ilgi vardır. Akustik travma<sup class="xref">3</sup><sup>,</sup><sup class="xref">4</sup><sup>,</sup><sup class="xref">5</sup>, ilaç<sup class="xref">6</sup>, hastalık<sup class="xref">7</sup><sup>,</sup><sup class="xref">8</sup>ve yaşlanma<sup class="xref">9</sup> maruz tüm Engelli işitsel işlevi HC ölüm ile sonuçlanabilir. Memelilerde saç hücre kaybı kalıcı yaralanma<sup class="xref">10</sup>' dan sonra yeniden oluşturabilirsiniz kuş HCs aksine.</p>
<p class="jove_content">Çağdaş araştırma çabalarının çeşitli çalışmışlardır kayıp HCs, geri yüklemek belirli deneysel yaklaşımlar farklılık. Duyu epiteli gen ifadesinde manipülasyon ve vücut dışında farklı kök hücre implantasyonu baskın yaklaşım alanında, kök hücre koklear organoids ayırt etmeye indüksiyon yöntemleri olmasına rağmen vardır <sup class="xref">11</sup><sup>,</sup><sup class="xref">12</sup><sup>,</sup><sup class="xref">13</sup>çalıştı. Her bu yaklaşımların olması güvenen doğrudan kök hücre veya kök hücreleri tarafından; kullanılan gelişimsel ipuçları Ancak, bir saniye paylaşılan ve potansiyel olarak kritik koklea kendisi ECM öğedir<sup class="xref">14</sup><sup>,</sup><sup class="xref">15</sup>.</p>
<p class="jove_content">ECM sadece fiziksel için hücre ve doku, hücre adezyon, nükleer silahların yayılmasına karşı hayatta kalma ve geçiş için bir yüzey içerir, ancak aynı zamanda HCs ve sarmal ganglion<sup class="xref">15</sup><sup>,</sup><sup class="xref">16 gelişiminde önemli roller oynar destekler </sup> <sup>,</sup><sup class="xref">17</sup>. Doğal olarak meydana gelen ECM hücre fenotip belirlenmesi ve/veya hücre adezyon, yayılmasını önleme ve hayatta kalma<sup class="xref">18</sup>rehberlik endüktif sinyalleri sağlar. Sonuç olarak, kültürlü hWJCs ile birlikte decellularized koklea kullanımı ECM ve HC rejenerasyon rol keşfetmek için eşsiz bir fırsat sunuyoruz. HWJCs Mezenkimal Kök hücre<sup class="xref">19</sup>gibi davranan insan göbek bağları gelen izole bir hazır, tartışmalı olmayan hücre türü vardır. HWJCs neurosensory hücre soy<sup class="xref">20</sup><sup>,</sup><sup class="xref">21</sup>ayırt etmek için yetenek göstermiştir. Böylece, geçerli protokol izolasyon, decellularization ve cochleae C57BL fare leşleri ile iç kulak doku mühendisliği için hWJCs dan perfüzyon ayrıntıları.</p>

<table><tbody><tr><td>Allegra X-14R Centrifuge</td><td>Beckman-Coulter</td><td>B08861</td><td></td></tr><tr><td>Intramedic Semi-Rigid Tubing</td><td>Becton Dickinson</td><td>427401</td><td></td></tr><tr><td>New Brunswick Innova 2000 Orbital Shaler</td><td>Eppendorf</td><td>M1190-0002</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14060-10</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools</td><td>11370-40</td><td></td></tr><tr><td>Ultra-Fine Forceps</td><td>Fine Science Tools</td><td>18155-13</td><td></td></tr><tr><td>50-mL Conical Tubes</td><td>Fisher Scientific</td><td>12565271</td><td></td></tr><tr><td>Petri Dish</td><td>Fisher Scientific</td><td>FB087579B</td><td></td></tr><tr><td>U-100 Insulin Syringe</td><td>Fisher Scientific</td><td>14-829-1B</td><td></td></tr><tr><td>Scintillation Vial</td><td>Fisher Scientific</td><td>03-341-73</td><td></td></tr><tr><td>Rotator</td><td>Fisher Scientific</td><td>88-861-049</td><td></td></tr><tr><td>Transfer Pipette</td><td>Fisher Scientific</td><td>22-170-404</td><td></td></tr><tr><td>Razor Blade</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Fisher Scientific</td><td>15-240-062</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Fisher Scientific</td><td>15-140-122</td><td></td></tr><tr><td>24-Well Plate</td><td>Fisher Scientific</td><td>07-200-84</td><td></td></tr><tr><td>SuperFrost PLUS Glass Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Transfer Pipette</td><td>Fisher Scientific</td><td>22-170-404</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Fisher Scientific</td><td>P36935</td><td></td></tr><tr><td>Clear-Rite 3</td><td>Fisher Scientific</td><td>22-046341</td><td></td></tr><tr><td>Thermo Scientific Forma Series II 3110 Water-Jacekted CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher Scientific</td><td>13-998-078</td><td></td></tr><tr><td>Mesenchymal Stem Cell Growth Medium</td><td>Lonza</td><td>PT-3001</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Lonza</td><td>CC-3232</td><td></td></tr><tr><td>TPP T-75 Culture Flask</td><td>MidSci</td><td>TP90076</td><td></td></tr><tr><td>TPP T-150 Culture Flask</td><td>MidSci</td><td>TP90151</td><td></td></tr><tr><td>TPP T-300 Culture Flask</td><td>MidSci</td><td>TP90301</td><td></td></tr><tr><td>Dissection Microscope</td><td>Nikon Instruments</td><td>SMZ800</td><td></td></tr><tr><td>Nikon Eclipse Ts2R-FL Inverted Microscope</td><td>Nikon Instruments</td><td>MFA51010</td><td></td></tr><tr><td>NuAire Class II, Type A2 Biosafety Cabinet</td><td>NuAire</td><td>NU-425-600</td><td></td></tr><tr><td>1X PBS</td><td>Sigma-Aldrich</td><td>P5368-10PAK</td><td></td></tr><tr><td>1% SDS Solution</td><td>Sigma-Aldrich</td><td>436143-100G</td><td></td></tr><tr><td>10% EDTA</td><td>Sigma-Aldrich</td><td>E9884-100G</td><td></td></tr></tbody></table>

a protocol for decellularizing mouse cochleae for inner ear tissue engineering

<p class="jove_content">Memelilerde, işitme eksikliği yeniden yeteneği kolaylaştırmak mechanosensory saç hücreleri olan işitme kaybı için tedavi sınırlıdır. Geçerli rejeneratif tıp stratejileri kök hücre veya destek hücreleri değiştirme hasarlı kök hücrelerin işitme kaybı düzeltmek için teşvik etmek için iç kulak çevresindeki genetik manipülasyon nakli üzerinde odaklanmıştır. Henüz, hücre dışı Matriks (ECM) inducing ve saç hücreleri işlevini sürdürmek hayati bir rol oynayabilir ve değil de soruşturma. Koklear kullanarak nasıl kompozisyon ve ekstraselüler ortamın mimari AIDS hücreleri işitme fonksiyonu sürdürülmesi içine benzersiz anlayışlar ECM bir iskele olarak erişkin kök hücreleri büyümeye sağlayabilir. Burada izole ve cochleae üzerinden periosteum erişkin kök hücreleri kabul iskele kullanmak için fare decellularizing bir yöntem mevcut. Geçerli protokol cochleae decellularized ve decalcified euthanized fare, izole edilmiştir. Daha sonra göbek kordonu izole edildi insan Wharton'ın jöle hücreleri (hWJCs) dikkatle her koklea derin. Cochleae Biyoreaktörler kullanıldı ve analiz için işleme geçiren 30 gün için hücreler kültürlü. Decellularized cochleae olarak tanımlanabilir hücre dışı yapılar korunur ama hücrelerin varlığı veya DNA'ın dikkat çeken parçaları olmadığını göstermiştir. Koklea derin hücreler içte ve dışta koklea çoğunu işgal ve olay olmadan 30 gün süre içinde büyüdü. Böylece, geçerli yöntemi nasıl koklear ECM etkiler hücre gelişimi ve davranış çalışma için kullanılabilir.</p>

<p class="jove_content" fo:keep-together.within-page="1">Burada sunulan yöntemleri kullanarak, cochleae başarılı decellularization varlığı ya da yokluğu DNA ile 4', inceleyerek değerlendirildi 6-diamidino-2-phenylindole (DAPI) boyama. DNA decellularized koklea içinde tanımlanmamış Eğer cochleae tam olarak decellularized kabul edildi. Yerel bir koklea decellularization veya incelemelerine tabi değil bir önceki deneme olumlu bir denetim yapıları ve geleneksel olarak bir C57BL fare koklea içinde bulunan hücreleri göstermek için kullanıldı. İnfüzyon herhangi bir hWJCs yoktu, decellularized-decalcified koklea, bir negatif kontrol kullanıldı. Hiçbir ölçülebilir lekeli DAPI hücre çekirdeği doku bölüm (<strong class="xfig">şekil 3</strong>) herhangi bir bölgede tespit edildi. İki cochleae decellularized decalcified ve sonra hWJCs geçerli proje için ilham. HWJCs ile derin decellularized-decalcified cochleae çok sayıda DAPI için çekirdek koklea ve koklea (<strong>Tablo 1</strong>) dışında kemikli kabuk üzerinde büyüyen içinde lekeli tespit edildi. Toplam tahmini hücre yoğunluğu hücre için infüzyon ilk koklea vardı 113,759 hücreler/koklea sonra kültür (<strong class="xfig">şekil 4</strong> ve <strong class="xfig">şekil 5</strong>) ve ikinci koklea için 30 gün 30 gün sonra kültür 118,732 hücreler/koklea (<strong class="xfig">şekil 6</strong> ve <strong class="xfig">Şekil 7</strong>). Bu hücre yoğunluğu tahminler Scala tympani, Scala medya ve Scala antre ve hacimli üç sıvı boşluk içinde bulunan yapılar içerir, ancak bu tahminler herhangi bir kalan kemik labirent hariç. Buna ek olarak, her koklea örnekleri Hematoksilen ve Eozin ile lekeli (H &#38; E) hücreleri ve anatomik özellikleri her koklea (<strong class="xfig">şekil 8</strong>) içinde varlığını göstermek için. Koklear microstructures gross anatomi tüm lekeli bölümleri boyunca ağırlıklı olarak değişmeden kalmıştır; Ancak, hiçbir hücre decellularized decalcified bölümünde (<strong class="xfig">şekil 8B</strong>) olarak tanımlanabilir. Hücre çekirdeği tanımlaması önemli ölçüde farklı arada yerel koklea (<strong class="xfig">şekil 8A</strong>) ve infüzyon içeren hücreleri (<strong class="xfig">şekil 8 c</strong><strong>-</strong><strong>D</strong>) decellularized-decalcified cochleae.</p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56523/56523fig1.jpg"><br>
<strong>Şekil 1: fare temporal kemik yalıtım.</strong> Fare uygun şekilde euthanized sonra keserek kafatası tabanı ve ilk servikal omur arasında hayvan başını kesmek ve kafa<strong>(a)</strong>dezenfekte etanol ile sprey. Cerrahi makas, beyin doku ve kemik ile kesme keskin bir çift kullanarak kafatası (<strong>B</strong><strong>-</strong><strong>C</strong>) bölümünün bir sagittal düzlemde bisect. Fare böldüğü kafa (<strong>D</strong>, alt çene için ilişkisiz deneysel iş kaldırıldı) o zaman temporal kemik ortaya çıkarmak için kaldırıldı beyin doku olması gerekir. İki foramina üzerinden işitsel ve vestibular sinir pass (<strong>E</strong>, kırmızı ok uçları) yararlı ipuçları başarıyla temporal kemik tanımlamak için. Eti kafatasından (<strong>F</strong>) kaldırın. Dış kulak kanalının (<strong>F</strong>, ok, üst panel) yararlı bir harici işaret de sağlar. Dikkatlice uzakta sadece izole temporal kemik (<strong>G</strong>) bırakarak aşırı kemik kırpın. Bulla (<strong>H</strong>, üst panel, kırmızı vurgulanan alanı) sonra ince ince forseps (<strong>H</strong>, alt paneli) kullanılarak kaldırılmalıdır. Bulla ince kemik ve kemik labirent koklea ortaya (<strong>J</strong>, kırmızı parantez) kadar kolayca uzağa (<strong>Ben</strong>) yontma. Ölçek barlar tüm görüntüleri vardır 1 mm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56523/56523fig2.jpg"><br>
<strong>Şekil 2: koklear perfüzyon.</strong> Temporal kemik izole edildikten ve bulla kaldırıldıktan sonra koklea sonra mikroskop, perfüzyon için ayarlanabilir. El ile tahrik boru ile kullanarak Ventriküler (çizgi noktalı<strong>A</strong>,) bağlı bir çift kendi kendine kapanma forseps kullanarak koklea stabilize etmek yararlı olabilir. Yavaşça forseps temporal kemik (<strong>B</strong>) Vestibüler parçası takın ve forseps Petri kabına<strong>(a)</strong>dudak karşı dinlenmek. Bu iki el aletleri perfüzyon sırasında işlemek özgür bırakır. Bir yandan sonra Öte yandan tüp oval forseps (<strong>B</strong>) ikinci bir çifti kullanarak penceresi üzerinde konumlandırabilirsiniz iken sıvı, Debi kontrol şırınga, üzerinde değişiklik yapabilir. Ücretsiz tüp sonuna bir açıyla kesme, daha kolay konumlandırma için doğru görüş alanı engellenmeden yerleştirilecek tüp izin verir. Ölçek çubuğu olduğunu 1 mm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56523/56523fig3.jpg"><br>
<strong>Resim 3: bir decellularized fare koklea bölümünü doku.</strong> Dik bir epi-floresan mikroskobu örnekleri (10 X mercek ve 20 X objektif) 200 X büyütme, görüntü için kullanıldı. Görüntüleri birlikte yaklaşık 4 x 5 resim dizisi oluşturmak için dikişli. DAPI hücrelerin yokluğu gösteren boyama nükleer DAPI ortak bir filtre set (350 nm uyarma, 470 nm emisyon) ve pozlanmış bir kullanarak panel <strong>A </strong>floresein isothiocyanate (FITC) genel filtre kullanarak autofluorescence gri tonlamalı görüntü gösterilir Anatomik yerlerinden sağlayan küme (490 nm uyarma, 525 nm emisyon), <strong>B</strong>panelinde gösterilir. Her iki panelleri <strong>A</strong> ve <strong>B</strong>belirlenen faiz, üç bölgelerinde otomatik hücre sayımları yapıldı. Toplam hücre sayımları tüm doku bölümü (<strong>1</strong>, düz beyaz çizgi) ve koklea (<strong>2</strong>, kesikli çizgi) için hesaplanır. Bu iki sayı koklea dışında ama doku bölümü (<strong>3</strong>, noktalı çizgi ve kesikli çizgi arasındaki boşluk) dış, kemikli kenarlarını içinde kemik yapılarda hücre sayısını hesaplamak için kullanılmıştır. Ölçek barlar tüm görüntüleri 500 µm vardır.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a><p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56523/56523fig4.jpg"><br>
<strong>Şekil 4: decellularized fare koklea 1 yakınındaki modiolar bir bölümünü infüzyon ile hWJCs.</strong> DAPI nükleer boyama <strong>A</strong> anatomik yerler sağlar yeşil kanal pozlanmış, bir gri tonlama autofluorescence üzerine bindirilmiş panelinde gösterilir. Hücre sayımları <strong>A</strong>panelinde belirlenen faiz, üç bölgelerinde gerçekleştirilen otomatik. Toplam hücre sayımları tüm doku bölümü (<strong>1</strong>, düz beyaz çizgi) ve koklea (<strong>2</strong>, kesikli çizgi) için hesaplanır. Bu iki sayı koklea dışında ama doku bölümü (<strong>3</strong>, noktalı çizgi ve kesikli çizgi arasındaki boşluk) dış, kemikli kenarlarını içinde kemik yapılarda hücre sayısını hesaplamak için kullanılmıştır. İzole DAPI boyama <strong>B</strong>panelinde gösterilir ve otomatik miktar sırasında kullanılan DAPI boyama eşik <strong>C</strong>panelinde gösterilir. Ölçek barlar tüm görüntüleri vardır 500 µm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig4large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56523/56523fig5.jpg"><br>
<strong>Şekil 5: decellularized fare koklea 1 modiolar bir bölümünü yüksek büyütme görünümünü infüzyon ile hWJCs.</strong> DAPI nükleer boyama <strong>A</strong> anatomik yerler sağlar yeşil kanal autofluorescence bir pozlanmış, gri tonlamalı görüntü üzerine bindirilmiş panelinde gösterilir. İzole DAPI boyama <strong>B</strong>panelinde gösterilir ve otomatik Hücre sayımı sırasında kullanılan eşik resim <strong>C</strong>panelinde gösterilir. Sıvı alanlarda ve koklea içinde yer alan ince microstructures güzel decellularization sırasında korunur ve deneme aşamaları kültür hücre. Scala Vestibuli (SV), Scala Tympani (SM), Basilar membran (BM), Tectorial Membrane (TM), Spiral Limbus (L), Rosenthal'ın kanal (RC), Modiolus (M), Spiral bağ (SL) ve çizikleri Vascularis (<strong>*</strong>). Reissner'ın membran açıkça doku bölümlerde tanımlı değil ve bunun sonucunda Scala medya değil açıkça tanımlanmıştır. Ölçek barlar tüm görüntüleri vardır 250 µm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig5large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56523/56523fig6.jpg"><br>
<strong>Şekil 6: yakınındaki modiolar bir bölümünü hWJCs ile infüzyon decellularized fare koklea 2.</strong> DAPI nükleer boyama Masası'a <strong>A</strong>gri tonlama autofluorescence anatomik yerler sağlar yeşil kanal üzerine bindirilmiş, gösterilir. Hücre sayımları <strong>A</strong>panelinde belirlenen faiz, üç bölgelerinde gerçekleştirilen otomatik. Toplam hücre sayımları tüm doku bölümü (<strong>1</strong>, düz beyaz çizgi) ve koklea (<strong>2</strong>, kesikli çizgi) için hesaplanır. Bu iki sayı koklea dışında ama doku bölümü (<strong>3</strong>, noktalı çizgi ve kesik çizgiler arasındaki boşluk) dış, kemikli kenarlarını içinde kemik yapılarda hücre sayısını hesaplamak için kullanılmıştır. İzole DAPI boyama <strong>B</strong>panelinde gösterilir ve otomatik miktar sırasında kullanılan DAPI boyama eşik <strong>C</strong>panelinde gösterilir. Ölçek barlar tüm görüntüleri vardır 500 µm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig6large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56523/56523fig7.jpg"><br>
<strong>Şekil 7: hWJCs ile infüzyon decellularized fare koklea 2 modiolar bir bölümünü yüksek büyütme görünümünü.</strong> DAPI nükleer boyama Masası'a <strong>A</strong>gri tonlama autofluorescence anatomik yerler sağlar yeşil kanal üzerine bindirilmiş, gösterilir. İzole DAPI boyama <strong>B</strong>panelinde gösterilir ve otomatik Hücre sayımı sırasında kullanılan eşik resim <strong>C</strong>panelinde gösterilir. Sıvı alanlarda ve ince microstructures koklear içinde yer alan deneme decellularization ve hücre kültür aşamalarında korunur. Scala Vestibuli (SV), Scala Tympani (SM), Basilar membran (BM), Tectorial Membrane (TM), Spiral Limbus (L), Rosenthal'ın kanal (RC), Modiolus (M), Spiral bağ (SL) ve çizikleri Vascularis (<strong>*</strong>). Reissner'ın membran açıkça doku bölümlerde tanımlı değil ve bunun sonucunda Scala medya değil açıkça tanımlanmıştır. Ölçek barlar tüm görüntüleri vardır 250 µm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig7large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56523/56523fig8.jpg"><br>
<strong>Şekil 8: Hematoksilen ve Eozin kontrol ve tedavi cochleae boyama.</strong> Hematoksilen ve Eozin bölümleri mevcut yerel hücrelerle tipik bir koklea görüntüleme lekeli <strong>A</strong>panelinde gösterilir. Panel <strong>B</strong> panel <strong>A</strong>olarak aynı yapıları içerir, ancak hücre dışı matriks bırakır bir tam decellularized koklea edinilebilir. HWJCs ile infüzyon iki daha önce gösterilen cochleae <strong>C</strong> ve <strong>D</strong>panellerinde görülmektedir. Her ne kadar belirli hücre popülasyonlarının ve mekanlar önemli ölçüde değişmiş brüt koklear anatomi decellularization ve hücre kültür süreçleri ile ağırlıklı olarak değişmeden kalır. Scala Vestibuli (SV), Scala Tympani (SM), Basilar membran (BM), Tectorial Membrane (TM), Spiral Limbus (L), Rosenthal'ın kanal (RC), Modiolus (M), Spiral bağ (SL) ve çizikleri Vascularis (*). Ölçek barlar tüm görüntüleri vardır 250 µm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig8large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a></p>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td><strong>A1 Koklea + hücre</strong></td>
			<td><strong>A2 Koklea + hücre</strong></td>
			<td><strong>B2 negatif.</strong></td></tr></tbody></table>Denetim koklea
		
		<tr>
			<td><strong>Dış kemik labirent</strong></td>
			<td>3,758</td>
			<td>549</td>
			<td>0</td>
		</tr>
		<tr>
			<td><strong>Kemik dış koklea alanlarda</strong></td>
			<td>248</td>
			<td>586</td>
			<td>0</td>
		</tr>
		<tr>
			<td><strong>Koklea içinde</strong></td>
			<td>518</td>
			<td>701</td>
			<td>0</td>
		</tr>
		<tr>
			<td><strong>Toplam hücreleri</strong></td>
			<td>4,524</td>
			<td>1,836</td>
			<td>0</td>
		</tr>
		<tr>
			<td><strong>Bölüm birimi (μl)</strong></td>
			<td>0.0077</td>
			<td>0.0100</td>
			<td>0.0147</td>
		</tr>
		<tr>
			<td><strong>Koklea hacminin yüzde</strong></td>
			<td>%0,46</td>
			<td>%0.59</td>
			<td>%0,87</td>
		</tr>
		<tr>
			<td><strong>Hücre yoğunluğu koklea içinde tahmini</strong></td>
			<td>113,759</td>
			<td>118,732</td>
			<td>0</td>
		</tr>
	

<p class="jove_content" fo:keep-together.within-page="1"><strong>Tablo 1: hücre sayımları.</strong> Koklear bölümleri lekeli DAPI thresholded ve ImageJ (kemik labirent, temporal kemik koklear sıvı alanlarda dışında ve koklea içinde dışında) ilgi üst üste 3 bölgelerde otomatik sayım araçlarını kullanarak quantified vardı. Kesit koklear alanı da ImageJ içinde ölçüldü ve bu değer bölümü kalınlığı (10 µm) ile birlikte bölüm birimi hesaplamak için kullanıldı. Santi <em>ve ark.</em> tarafından Yayınlanan toplam koklear hacim tahmini kullanarak <sup class="xref">23</sup>, belirli bir bölüm göreli koklear hacmi hesaplanır. Tüm koklea hücre yoğunluğu eşlenmiş hücre sayısı ile birlikte verilen bir bölümünün göreli koklear birim için tahmin edilmiştir.</p>

Watch this Scientific Journal Video about A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering at JoVE.com

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Fare Cochleae iç kulak doku mühendisliği için Decellularizing için bir iletişim kuralı

<p class="jove_content">Bu iletişim kuralının amacı decellularize ve fare cochleae doku mühendisliği uygulamaları için iskele olarak kullanımı için decalcify için etkili bir yöntem göstermektir.</p>

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

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Bioengineering

10.7K Görüntüleme.  University of Kansas Medical Center. Bu iletişim kuralının amacı decellularize ve fare cochleae doku mühendisliği uygulamaları için iskele olarak kullanımı için decalcify için etkili bir yöntem göstermektir.

Video: A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Evolutionary Relationships

<p>Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4<sup>th</sup> century BC. Aristotle&#8217;s system was improved upon during the Renaissance and then, subsequently, by Carolus Linnaeus in the mid 1700&#8217;s. These more formal classification and organization systems grouped species by their physical similarity to one another. For example, all vertebrates have a backbone, but invertebrates do not. Traits like the backbone are called synapomorphies, which are traits that are shared by a group of organisms, presumably because they were derived from a common ancestor. As we will explore, this method has been shown to have limitations and has more recently been amended to include genetic analysis. Still, scientists construct trees called dendrograms to create a visual representation of how species are related to one another and share common ancestors. These dendrograms can aid in our understanding of the evolutionary processes that drive these relationships. Genetic comparisons have added an important tool guiding the analysis of evolutionary relationships.</p>
<h4>Types of Dendrogram</h4>
<p>A type of dendrogram, called a cladogram, depicts the hypothetical genealogical relationships between species with the tips (or leaves) of the tree representing a species and the branches showing how species are related to each other. A slightly more complicated type of tree, called a phylogram, differs from a cladogram in that the branches leading to the species are of different lengths. The length of a branch in this type of tree represents the degree of change between species: the longer the branch, the more time since the species have diverged from a common ancestor. In both types of tree, the common ancestor of a group of species is indicated by a node, which is the point where a series of branches meet. Species that are more closely related to each other (most recently shared a common ancestor) will be located closest to the node. The two species that share a node are called a sister group<sup>1</sup>.</p>
<h4>Understanding Evolutionary Relationships using Genetic Data</h4>
<p>Historically, cladograms were constructed by comparing the morphology (physical structure) of organisms. This method is still practiced but the techniques have been modernized to include comparison of DNA (deoxyribonucleic acid) sequences between species. Using DNA for building trees has several advantages over relying solely on morphology, including being able to calculate an estimate of how long ago different species shared a common ancestor<sup>1</sup>. However, using DNA is not always feasible, especially when trees include extinct organisms. DNA is best found in soft tissues, which are not preserved during the fossilization process, and therefore it is uncommon for a DNA sample of an extinct species to be available.</p>
<p>DNA is passed on from parents to their offspring in hereditary units called genes. The nucleotide (A, G, C, and T) sequence of genes found in different species are frequently quite similar, likely due to their having come from a common ancestor. This fact allows researchers to align sequences from different species with one another to build the trees described above. Species with more similarity between their nucleotide sequences will be placed next to each other in a tree, and species with less sequence similarity will be placed further apart from each other.</p>
<p>Bioinformatics are the tools used by biologists to analyze large datasets using a combination of computer science, mathematical modeling, and statistics. One such tool is called BLAST (Basic Local Alignment Search Tool), which can be used to quickly search the entire genome of any species that is available in the NCBI (National Center for Biotechnology Information) database<sup>2</sup>. The NCBI database combines several different databases that hold different types of DNA sequence information. The process of a BLAST search includes complex computer algorithms, but basically, BLAST aligns sequences of each nucleotide base from a submitted DNA sequence (known as the <em>query</em> sequence) with sequences in the data base that most closely match it. The DNA sequences that are found will be listed in order of similarity to the sequence in question, and will therefore be from species closely related to the species containing the query gene. This comparison may or may not depict the actual evolutionary relationship between species because genes evolve at different rates. Additionally, genomes sometimes contain more than one instance of a similar sequence.</p>
<p>Comparison of DNA sequences of genes is valuable beyond consideration of evolutionary relationships. Frequently, genes are identified in model organisms, such as the fruit fly, <em>Drosophila melanogaster</em>, or the mouse<sup>3</sup>. Integral to studying a gene, the function of its product is commonly identified and analyzed. If a researcher is interested in studying that function in a different organism (humans for example), BLAST or other bioinformatic tools can be used to find candidate genes based on their similarities to the genes of known function from model organisms.</p>
<p>Human genes can also be used as the starting point to find homologs in model organisms. In fact, human disease research depends heavily on this. Once a human gene of interest is identified, mice can be genetically manipulated to have the homologous gene disrupted, or &#8220;knocked out,&#8221; creating a model of the human disease that can be studied in order to understand and treat the disease. There are many of these mouse strains currently available. For example, there is a mouse model for human Cystic Fibrosis (CF) called the <em>Cftr</em> knockout mouse and another modeling atherosclerosis, called the <em>Apoe</em> knockout<sup>3</sup>.<br />
 </p>
<h4>References:</h4>
<ol class="references">
	<li><strong>Annenberg Learner.</strong> Online Textbook: Unit 3 Evolution and Phylogenetics. <em>Rediscovering Biology. </em>[Online] 2017. [Cited: August 23, 2018.] https://www.learner.org/courses/biology/textbook/compev/compev_3.html.</li>
	<li><strong>National Center for Biotechnology Information, U.S. National Library of Medicine.</strong> BLAST: Basic Local Alignment Search Tool. [Online] https://blast.ncbi.nlm.nih.gov/Blast.cgi.</li>
	<li><strong>National Human Genome Research Institute.</strong> Background on Mouse as a Model Organism. <em>National Human Genome Research Institute. </em>[Online] December 2002. [Cited: August 23, 2018.] <a href="https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/" target="__blank">https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/</a>.</li>
</ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Concept

Evrimsel İlişkiler

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system ...

Education

JoVE Lab Manual

Biyoloji

Evrim

Extraction

Extraction: Liquid-Liquid and Acid-Base Extraction - Procedure

Çıkarma

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Cellulose RecoveryExpand
	Extraction and filtration can ...

Kimya

Lab Manual Sub Articles

Electrolytic Cells

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Electrolytic Cells<button class="expand">Expand</button>
	<p>In this experiment, you will be assembling an electrolytic cell to perform electroplating. An electrolytic cell contains an anode and cathode connected by a power source, which drives the nonspontaneous redox reaction in the cell. When the anode material is oxidized, the ions travel to the cathode, where they are reduced and plated.</p>
	<p>In the case of this experiment, the electrolytic cell contains a copper anode and a brass key for the cathode. Oxidation of the anode creates copper(2+) ions, which are then reduced on the brass key, forming a thin plating of solid copper.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on gloves, safety goggles, and a lab apron, which must be worn at all times.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Obtain one 10-ohm resistor, one copper sheet electrode, one copper wire, and one piece of emery paper.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use the emery paper to polish the brass key and the copper sheet. Rinse the key and the copper electrode with water. The brass key will be the cathode, and the copper sheet will be the anode.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Take one 50-mL beaker and a watch glass to the instructor&#39;s hood and obtain approximately 20 mL of acetone. Place the watch glass on top of the beaker to prevent evaporation and bring it back to your bench.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Label the other 50-mL beaker as &#39;organic waste&#39;.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use a disposable pipette to rinse the copper electrode and key with acetone. Collect excess in the waste beaker. Place the key and copper electrode on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Measure the initial masses of the key and copper electrode and record their values in your lab notebook.
		<h4><strong>Table 1: Moles of Cu Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td></td>
					<td style="text-align: center;"><strong>Mass of key (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu plated </strong></td>
					<td style="text-align: center;"><strong>Mass of Cu electrode (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu lost </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">0 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/53/Table_1._Moles_of_Cu_Transferred.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set up the electrolytic cell. Use one electrical wire with alligator clips to connect the negative terminal on the battery to the current probe. Wrap the 10-ohm resistor around the other end of the current probe.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use another wire to connect the positive terminal on the battery to the copper electrode.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Wrap the copper wire around the brass key and use an electrical wire to connect the resistor on the current probe to the copper wire on the brass key.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Obtain about 200 mL of concentrated copper sulfate in a 250-mL beaker. The amount does not need to be exact. Bring the beaker back to your bench and set it on the stir plate. Carefully, place a magnetic stir bar in the beaker and turn the stir setting on to high.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Connect the data acquisition system to the current probe and allow the data acquisition system to sense the probe. It should return a reading in amps. Change the rate to two scans per min and the duration to 5 min.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Place the brass key in the beaker. Hang it off the side of the beaker so that the key is almost completely submerged but does not touch the magnetic stir bar. <strong>Note: </strong>The copper wire should not be in the solution.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Place the copper electrode in the beaker, bending it so that it hangs over the edge of the beaker. Ensure that it is not touching the stir bar or the brass key.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Start the scan and let it collect data for 5 min. This will act as a test run to make sure plating occurs on the brass key.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">After the scan ends, remove the key and copper electrode. The copper plating on the brass key should be evident. If it is not visible, check your circuit to make sure that the battery is connected in the correct orientation.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Once you have completed a successful 5-min plating, disconnect the key and copper electrode from the wiring. Remove the copper wire from the key and rinse the key and copper electrode with water and then acetone. Lay them on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Measure the masses of the key and the copper electrode and record them in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Reconnect the key and copper electrode and place them back in the solution like before. Restart the run on the data acquisition system, making sure that the rate is set to two scans per min. With the duration set to 30 min, allow the electroplating process to run.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">After the scan is complete, remove the electrode and key from the solution, disconnect them from the wires, and rinse them with water and then acetone. Allow them to dry on a paper towel, then weigh them and record their masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Reassemble the setup and repeat the electroplating for a second 30-min trial. When the scan is complete, remove the key and copper electrode, rinse them like before, and record their mass.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Save the recorded data as a text file on a flash drive.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">To clean up, turn the stir setting off and remove the stir bar from the solution using forceps. Dispose of the acetone in the organic waste container provided by your instructor. The copper sulfate solution can be reused for future labs, so pour the concentrated copper sulfate back into the carboy.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Rinse all of your glassware with water and return it to the instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">First, calculate the total charge Q that flowed through the system during the different trials. One coulomb is the quantity of electricity flowing through a wire carrying 1 ampere of current in 1 s, thus Q = I x t.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Review the current probe data for the first 5-min electroplating trial.<strong> Note: </strong>If the current dropped to 0 or close to 0 at the end of the trial, then the electrons stopped flowing from the copper electrode to the key. Only use the values recorded before the drop in current occurred. If the current drop to 0 early in the plating process, repeat the trial.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The current varies over time, so calculate the average current, I.
		<h4><strong>Table 2: Moles of Electrons Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>Time (s) </strong></td>
					<td style="text-align: center;"><strong>Current<sub>average</sub> (A) </strong></td>
					<td style="text-align: center;"><strong>Total charge (C) </strong></td>
					<td style="text-align: center;"><strong>Moles (e?) </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">Total</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/53/Table_2._Moles_of_Electrons_Transferred.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Use the average current over the total time to determine the total charge transferred during electroplating, Q. About 20 coulombs were transferred during the 5-min trial, and between 110 and 120 coulombs were transferred during each 30-min trial. The total charge transferred over the entire 65-min electroplating process was about 250 coulombs.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Use the total charge transferred to calculate the moles of electrons plated during each trial. The moles of electrons plated equals the total charge transferred divided by the Faraday constant. The Faraday constant represents the magnitude of electric charge per mole of electrons and is determined by multiplying the charge of an electron by Avogadro&#39;s constant.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Determine the moles of electrons that were transferred for the 5-min and 30-min trials.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the moles of copper that were plated on the key after each trial. Similarly, determine the moles of copper lost from the copper sheet electrode.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the moles of copper that were lost from the electrode to the amount that was plated to the brass key. The numbers should be close.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Check to see if these values match what is expected based on the number of moles of electrons that were transferred. The reduction of 1 mole of copper(2+) ions requires 2 moles of electrons to produce 1 mole of solid copper.</div>
		</li>
	</ul>
	</li>
</ol>

Electrolytic Cells: Construction and Electroplating - Procedure

Elektrolitik Hücreler

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Electrolytic CellsExpand
	In this experiment, you will be assembling an electrolytic cell to ...

Galvanic Cells

Galvanic Cells: Construction and Reduction Potential Measurement - Procedure

Galvanik Hücreler

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Constructing a Lead-Copper Galvanic CellExpand
	In this experiment, you will construct a ...

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing the Cell Cycle in a Root Tip
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Hypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a chemical that interferes with microtubular polymerization, all of the cells will be arrested at the same stage of the cell cycle and that in untreated onion tip slices all of the different stages of the cell cycle will be visualized. The null hypothesis is that there will be no differences in mitosis between the two groups and that different stages of the cell cycle will be visualized. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To prepare for this experiment you will use an onion that has been placed in water for several days until new white root tips have begun to sprout. Use a razor blade to cut a 1 cm piece from the end of one of the root tips. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the slice of onion into a microcentrifuge tube labeled &#8220;N&#8221; for the nocodazole condition. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, cut a second 1 cm piece of onion root tip and place in a microcentrifuge tube labeled &#8220;C&#8221; for the control condition.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Add 2 &#181;L of the 5 mg/mL nocodazole to 1 mL of water in the microcentrifuge tube labeled N and 1 mL of water only to the tube labeled C. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Leave the tubes containing the root tips to incubate for 12 hours and wipe down the workspace with 70% ethanol.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">When incubation is complete, set a heat block to 60 &#186;C.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Wash the nocodazole treated tips with water and then aspirate the liquid.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Repeat this wash two more times, for a total of three washes. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Fill both tubes with 1 mL of one normal hydrochloric acid. Incubate the tubes in a 60 &#186;C heat block for 12 - 15 min, then discard the acid into a waste flask.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Now, add 1 mL of distilled water drop wise to the tubes at least three times to rinse the samples.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Now add one microliter of DAPI stain to 10 mL of phosphate buffered saline (PBS).
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then place 50 &#181;L of this dilute DAPI stain into each tube, and leave them at room temperature to incubate for 12 min. 
</div></li><li data-sub-note-item="1-14"><div class="lab-step">After this, preform three distilled water washes as in step 10. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Label one microscope slide as 'Control' and another as 'Nocodazole'. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Transfer the stained roots onto their respective microscope slides and add a drop of water to the control slide and a drop of nocodazole to the nocodazole slide.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Use the razor blade to remove the unstained portions of each root tip and place a glass cover slip over each sample.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Carefully press down on each cover slip with a gloved fingertip, and then let the slides sit for 10 min. 
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Finally, view the slides under the 40X objective of a fluorescence microscope and capture images of your root tip cell preparations.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Understanding the Loss of Cell Cycle Control
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: Before cells can pass through all of the stages of the cell cycle there are several checkpoints they must go through. These checkpoints are regulated by cyclin and cyclin-dependent kinase proteins, or CDKs. If the cyclins and CDKs determine that previous cellular events have not been adequately completed they will arrest the cells at this step and prevent them from proliferating. In cancer cells one or several of these molecular restraints for regulating cell division are lost and this allows the damaged cells to escape from cell proliferation control. These abnormal cells develop into tumors which are masses of uncontrolled proliferating cells that interfere with the normal functions of the body. Notice how before it divides this cancer cell rounds up and refracts light very strongly under the light microscope. This is because not all cancer cells successfully divide into daughter cells after each round of mitosis. This will mean that they often contain an excess of organelles and DNA and this increase in cellular content means they will refract light more intensely than normal cells do. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Examine your collected images and record in Table 1 the different cell structures and stages that could be identified in the onion tip slice that was treated with nocodazole. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/10/Table_1_Cell_division.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">If there are any cells undergoing mitosis, note what stages of cell division you see.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Record the percentage of cells in each stage of mitosis in Table 1. 
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Next, examine the images of the untreated onion tip slice, and record what cell structures can you identify. 
</div></li><li data-sub-note-item="3-5"><div class="lab-step">If there are any cells undergoing mitosis, consider what stages of cell division you see, and record the percentages of cells in each stage of mitosis in the Table 1. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Procedure

Hücre Bölünmesi

Observing the Cell Cycle in a Root Tip
ExpandHypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a ...

Hücre süreçleri

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Human Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this experiment you will prepare different types of cells from a plant and animal, onion and human respectively, and then visualize them under a microscope. HYPOTHESES: The experimental hypothesis is that the cells will appear different in overall shape and membrane structure, but there will be some shared similar structures, like nuclei, in both cells. The null hypothesis of the experiment is that there will be no difference in structure between the two cell types.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">First, to prepare an animal cell slide, start by pouring 30 mL of distilled water into a beaker.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, use a glass dropper to place 2 - 3 drops of the water onto the center of a clean microscope slide.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, take a clean toothpick and scrape the inside of your cheek to gather cells.  CAUTION: Do not scrape your cheeks too aggressively. The scraping should not cause discomfort or bleeding. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Put the end of the toothpick with the cheek cells onto the wetted area of the slide and mix the water and cheek cells together.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, add two drops of methylene blue staining solution to the wetted area of the slide and mix it in with a clean toothpick.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Let the mixture sit at room temperature for 3 minutes.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After 3 minutes have passed, use a glass dropper to add a drop of glycerol to the mixture.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Carefully, place a cover slip over the mix on the slide by first standing one end of the cover slip vertically and aligning it along to one side of the mixture. Then, carefully lower the other end of the cover slip down until the mixture is completely covered.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Finally, remove any excess liquid with blotting paper.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Next, prepare a slide of plant cells by placing five drops of distilled water into a clean watch glass.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, with forceps, take a thin strip of onion and place it into the water.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Apply 5 drops of safranin solution to another watch glass.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Now using a pair of forceps transfer the piece of onion from the distilled water into the safranin.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the onion to soak for 30 seconds.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, again with forceps, move the onion back into the distilled water.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, place 3 drops of glycerol onto the center of a glass microscope slide.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Using the forceps again, transfer the onion into the glycerol on the slide.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Gently place a cover slip over the onion on the slide and blot away any excess liquid with a piece of blotting paper.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you will visualize the slides you have prepared using a compound microscope. First, turn on the scope and set the objective lens to the lowest level of magnification.  NOTE: A compound microscope typically has two levels of magnification.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Place the cheek tissue slide onto the stage of the microscope and bring the cells into focus by adjusting the stage and using the coarse adjustment knob.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">When the cells are in focus, observe the individual cells and draw what you see. Repeat these observations at medium and high magnifications adjusting the focus with the fine adjustment knob. IMPORTANT: Do NOT use the coarse adjustment knob at high magnifications. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, rotate the nose piece so that the oil lens is almost in place.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Place a drop of immersion oil on top of the cover slip of the cheek cell slide.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Use the oil lens to view the cheek cells and record your observations.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Repeat these steps to view the onion plant cell, moving again from low to medium, high, and oil lenses (steps 20 &#8211; 24).
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record all of your observations and note the differences you see between animal and plant cells.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">When you are finished, place the cheek cell slide and toothpicks into a diluted bleach solution. Dispose of the cover slips into a glass waste container.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Finally, turn off the microscope and clean the lenses with lens cleaning paper.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Calculate the total magnification for each of the viewing levels. Remember to account for both the eyepiece magnification and the objective lens magnification. NOTE: Eye piece: 10X magnification, Objective lens: 4X, 10X, and 40X magnification
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, identify the structural differences between plant and animal cells using the observations made under the microscope.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Label the nuclei in both cell types and then highlight or note any differences that you see between them.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Procedure

Hücre Yapısı

Visualizing Onion and Human Cells
ExpandIn this experiment you will prepare different types of cells from a plant and animal, onion and human ...

Temel Bilgiler

Genetics of Organisms

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">REF and WEF Fly Genetics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be labeled with the notation REF for red-eyed flies or WEF for white-eyed flies. The female vial should also be labeled virgin as these flies should be unmated for the purpose of this experiment.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Make sure you have around five flies in each vial. NOTE: You will perform crosses using these flies to determine if red or white eyes is dominant and if the trait is sex-linked. You should also have two fly anesthesia wands, a paint brush, magnifying glass, fly anesthetic, some empty fly vials, and a sheet of white paper.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Draw a line in the middle of the paper to make a male side and a female side.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Dip the fly anesthesia wand into the solution and insert it into the red-eyed female vial until the flies stop moving.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Then, dip the same wand into the white-eyed male vial until these flies are also anesthetized.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Tap the flies from the two vials onto the sheet - Females on one side and males on the other.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Check to make sure that all the flies from the female vial are female and likewise, confirm that the male flies are male.  NOTE: Males are slightly smaller and have a darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If there are any male flies in the female vial, obtain a new vial as these females likely have sperm from males from their own strain and are unusable.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Now, move the five red-eyed females and five white-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Label this vial to indicate that this is a cross between red-eyed females and white-eyed males. HYPOTHESES: Use Punnett squares to formulate hypotheses for this experiment. Consider what you will see in a few days if the white allele is dominant and there is no effect of parental sex. What if red is dominant? Under the potential offspring genotype in each box, write the expected phenotype.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">To set up the reverse cross, again dip the fly anesthesia wand into the solution and insert it this time into the white-eyed female vial and then the red-eyed male vial until all the flies have stopped moving.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Tap the flies onto a sheet of white paper and check to make sure that all the flies from the female vial are female and likewise with the male flies.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Now, move the five white-eyed females and five red-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Label this vial to indicate that this is a cross between white-eyed females and red-eyed males.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, place the two tubes containing the new crosses into a rack for incubation at room temperature. NOTE: After about three days, larvae should be moving around in the vial.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Remove the 10 adult flies and dispose of them into the morgue tube containing 70% ethanol.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, put the tubes back in the rack and let the larvae mature until they reach the adult stage in up to two weeks.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Once all of the larvae from these crosses have matured to adulthood, anesthetize the flies in one of the tubes using the wand.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Then, on a piece of white paper, sort the flies by sex and phenotype and then record these data.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Now, repeat this process and tally the sexes and phenotypes for the second cross (steps 1 &#8211; 18).
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Finally, once you have counted all of your flies, dispose of them in the morgue vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin investigating your results, first compare the data you collected with the hypothesis you made using the Punnett squares.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Did your data match the simple Punnett square hypothesis where red or white is dominant and there is no effect of sex? Compare your data with other lab groups.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">If your data does not match what your Punnett squares predicted, does this indicate that the gene may be sex-linked?
</div></li><li data-sub-note-item="2-4"><div class="lab-step">If the red gene were dominant and sex-linked, when we cross red-eyed females with white-eyed males, all of the offspring would have red eyes. Assuming all of the females were homozygous for the color allele, does this match your data for the first cross?
</div></li><li data-sub-note-item="2-5"><div class="lab-step">If white were dominant and the gene were sex-linked, we would see the exact opposite. So all of the flies would have white eyes. From your data, can you conclude from your first cross whether red or white is dominant?
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When the reciprocal cross of white-eyed females and red-eyed males is performed, if the red phenotype is dominant, all of the females would be red and all of the males would be white. This would also confirm that the gene is specifically x-linked because white-eyed females could then only be produced in crosses with white-eyed males. Did your second cross produce any white-eyed females?
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Finally, if the gene were x-linked and white was dominant, crossing white-eyed females with red-eyed males would result in all of the offspring having white eyes. Does this hypothesis fit with your second cross?
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Look over your data and conclude which of the hypotheses best fits the patterns you found in your crosses. If your data does not fit with any of these, hypothesize why this might be the case.
</div></li></ul></li></ol>

Genetics of Organisms: REF and WEF Fly Genetics - Procedure

Organizmaların Genetiği

REF and WEF Fly Genetics
ExpandNOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be ...

Genetik

Microbial and Fungal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Examining Microbial Colony Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">As the colonies are identified, fill out the observed colony properties in the appropriate column in the table.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/28/Table_1_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, repeat the observations for each of the other two bacterial species.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Place two loopfuls of sterile distilled water onto the center of the slide.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, cover the slide with Gram&#39;s iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Decolorize the slide with 95% ethanol until stain no longer washes away.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Gently rinse the slide with distilled water and blot dry with absorbent paper.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After staining the other two slides in the same manner (steps 6 &#8211; 17), observe each with a microscope.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria&#39;s shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/28/Table_2_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Examining Fungal Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Remove the <em>S. cerevisiae</em> plate from the incubator and place a drop of water onto a microscope slide.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using a sterilized inoculating loop, smear a film of yeast into the drop of water and place a cover slip over the slide.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Place the slide under the microscope at the lowest magnification, increasing the magnification to the oil immersion setting to see the individual yeast cells.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Observe the general shape and structure of the yeast and identify any cells that are undergoing asexual reproduction by budding.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, draw the cells in the appropriate magnification circles.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When you are done observing the yeast, replace the <em>S. cerevisiae</em> slide with a prepared Ascomycetes apothecium slide and set the microscope to the lowest magnification.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using colored pencils, draw the structures at low and high magnifications, including the asci and ascospores.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Finally, place a button mushroom, <em>Agaricus bisporus</em>, under the dissecting microscope and draw the features, including the pileus, or cap, the gills, and the stipe, or stalk.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Using the coloring of the bacteria recorded in the table, determine which species are Gram-positive and which are Gram-negative.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, look at the shapes recorded in the table to determine which bacterial species are bacillus, coccus, or spirillum.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next compare your illustrations of the yeast cells to your illustrations of the bacterial cells. NOTE: Yeast from the Ascomycetes phylum, such as <em>S. cerevisiae</em>, can reproduce asexually, which can be observed through the budding of the cells under a microscope.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any observations of yeast cells budding.  NOTE: Ascomycetes can also reproduce sexually via mitosis and haploid spore formation.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Record any observations of spore or asci formation you observed in the table.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">NOTE: Fungus from the Basidiomycota phylum exhibit clearly observable gills under the pileus.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record any fungal species that demonstrated gills under the pileus.
</div></li></ul></li></ol>

Microbial Colony and Fungal Diversity - Procedure

Mikrobiyal ve Fungal Çeşitlilik

Examining Microbial Colony Structures
ExpandTo begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
Place ...

Community Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Measuring Biodiversity in Areas with Different Disturbance Regimes
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this experiment, you will compare the biodiversity in a less disturbed area against a more highly disturbed area. HYPOTHESES: The experimental hypothesis could be that a less disturbed area will contain a greater diversity of species than a highly disturbed area. The null hypothesis could be that there will be no difference in diversity between less disturbed and highly disturbed areas. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin, divide into groups of 2-4 and collect a hula hoop, ball, measuring tape, set of printed tables, 10 utility flags, and an ID guide from the front of the class.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Additionally, collect a shovel and 10 collecting bottles for the soil sampling exercise.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Once in the field, your instructor will show you the two habitats you will study - choose one habitat to begin.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Place one flag in each of four corners to form a rectangular sampling area.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Measure the length and width of the sampling area in meters, and then multiply the two numbers to determine the area.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Standing in the middle of the sampling area, throw a tennis ball into the air, and then Mark the location where it lands with a flag.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Repeat this random throw four additional times to determine all five of your random sampling points.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Keeping your flag at the center, place a hula hoop around it. The area within the hula hoop is a quadrat.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Identify all of the different species in the quadrat and record them as species A, B, and so on in Table 1, provided by your instructor.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once all of the plants have been recorded, use a shovel to collect one cup of soil from each of the sites and put the samples bottles labeled with your names and the site name and quadrat number.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Repeat the plant identification and soil sampling for the remaining four quadrats in the first habitat, recording your observations in Table 1.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Once you have completed recording in the first habitat, move on to the second field, again marking out a rectangular sampling area.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Repeat the random placement of quadrats using the tennis ball, and then record the species present in each quadrat in Table 2. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, collect one cup of soil from each of the disturbed habitats&#39; quadrats, for a total of five cups from the disturbed and five cups from the undisturbed habitats.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Measuring Biodiversity Along a Transect
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: The next part of the study will test for edge effects, or a change along a gradient from where one ecosystem type meets another to the core habitat of the ecosystem. HYPOTHESES: The experimental hypothesis is that there will be a greater diversity of species closer to the edge of the ecosystem than in the center, or core, of the ecosystem. The null hypothesis is that there is no change in diversity along the transect from edge to core habitat.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Place a flag at the perimeter of the field where it meets another habitat, like a forest or parking lot.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Extend a tape measure towards the center of the field, perpendicular to the edge of the field. This will be the transect.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Place a flag at the zero meter of the transect (the edge of the other habitat) then place additional flags at 10 m, 20 m, 30 m, and 40 m into the core of the habitat for a total of five flagged locations.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Starting at the 0 m point along the transect, place the hula hoop at the flagged location. Record the species present in the quadrat in Table 4.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Now repeat the quadrant placement and species recording at the 10 m, 20 m, 30 m, and 40 m marks.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Next, place two more transects from different starting points at the perimeter of the field extending into the core area.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Mark the 10-meter points along these transects, and again use these as markers to place quadrats. Record the species found in each of these quadrats in Table 4.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Processing the Soil Samples
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">HYPOTHESES: The experimental hypothesis is that the two landscapes under different disturbance regimes will differ in their soil composition and pH. The null hypothesis is that there will be no difference in the soil composition or pH between the two sites. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">To begin, remove soil from one bottle of the first site sampled and fill the pH color compartment of the soil sample kit to the soil fill line.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Pour the pH test capsule into the test chamber, then fill the capsule with distilled water until it reaches the water fill line.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Place the cap on the compartment and shake it.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Allow the color to develop for about a minute, and then compare the soil to the color chart to determine the pH.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Repeat the pH analysis for the second sampled site, filling out Table 3 for the recorded pH.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">To process the soil samples for nitrogen, phosphorus, and potassium, remove 20 mL of soil from the bottle of the first site sampled.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Place the soil in a clean container with 100 mL of distilled water.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Repeat this process for the second site, labeling each container, and then allow them to sit for one hour.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">After one hour, use a dropper to fill each of the nitrogen, phosphorous, and potassium comparators to the fill line with solution from the first sampled site.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">Add the appropriate capsule to each of the comparators for each chemical test.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">Cap and shake the comparators thoroughly, and then compare the solution in the test chamber to the color chart, recording the results as the appropriate number on the scale into Table 3.
</div></li><li data-sub-note-item="3-13"><div class="lab-step">Repeat the nitrogen, phosphorous, and potassium tests for each of the sampled sites. NOTE: Rinse the comparator thoroughly between samples
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">To calculate the Shannon-Weiner Diversity for each of the observed sites, first open a new Excel spreadsheet.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Label cell A1 'species name', and cell B1 'total count'.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Starting from the second row and in column A, fill in the sheet for each species found in the first site, using the data recorded in Table 1.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Calculate the total number of each species found in the entire sampling effort and record this in the B column of the Excel spreadsheet.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Now, label cell G1 'total count of all species', and in cell H1, write a formula, or highlight the appropriate cells to sum all of the count values entered in column B: = SUM(B2: BX)
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Label cell C1 'proportion of each species' and in cell C2, type the formula for the proportion of the total of each species at the site. = B2/H$1
</div></li><li data-sub-note-item="4-7"><div class="lab-step">Drag this formula down for each row of data represented in column B.
</div></li><li data-sub-note-item="4-8"><div class="lab-step">Label cell D1 'natural log proportion of species' and in cell D2, type the formula for the natural log of the proportion of species at the site. =LN(C2)
</div></li><li data-sub-note-item="4-9"><div class="lab-step">Drag this formula down for each row of data represented in column C.
</div></li><li data-sub-note-item="4-10"><div class="lab-step">Add the formula into cell E1 for the proportion of species multiplied by the natural log of the proportion of species: ps* ln ps
</div></li><li data-sub-note-item="4-11"><div class="lab-step">In cell E2, type the formula multiplying the proportion of species by the natural log of the proportion of species at each site, and then drag this formula down for each row of data represented in column D. = C2 * D2 
</div></li><li data-sub-note-item="4-12"><div class="lab-step">Label cell G2 as 'sum of ps * ln ps', and then, in cell H2, type a formula summing all of the values in column E. = SUM(E3 - EX) 
</div></li><li data-sub-note-item="4-13"><div class="lab-step">Label cell G3 'Shannon-Weiner Index', and in cell H3, enter the formula for the Shannon-Weiner Diversity Index. = (EXP - 1 * H2) NOTE: This is the calculated Shannon-Weiner Diversity Index for the first sampled site, and will be used for comparison between the two sites.
</div></li><li data-sub-note-item="4-14"><div class="lab-step">Repeat the data analysis in a new sheet using the data collected for the second site, which was recorded in Table 2. NOTE: Areas with greater index values are considered to have greater species diversity. Note whether you see differences between your sites.
</div></li><li data-sub-note-item="4-15"><div class="lab-step">Repeat the Shannon-Weiner Diversity calculations using a total count of all the species at each distance along the transect, then create a table of all of these values in a new Excel sheet.
</div></li><li data-sub-note-item="4-16"><div class="lab-step">Click on the Data tab in Excel, and then on the 'Data Analysis' button.
</div></li><li data-sub-note-item="4-17"><div class="lab-step">Select 'Regression' and click 'Okay'.
</div></li><li data-sub-note-item="4-18"><div class="lab-step">For the Input Y Range, highlight the Shannon-Weiner Diversity values, and for the Input X range, highlight the distance values.
</div></li><li data-sub-note-item="4-19"><div class="lab-step">Select an output location for the regression results and click 'Okay'.
</div></li><li data-sub-note-item="4-20"><div class="lab-step">Check the P-value box in the intercept row to see if there is a significant correlation between distance from edge to core habitat and biodiversity. NOTE: A value of less than 0.05 rejects the null hypothesis of no change in diversity along the transect, while a value greater than 0.05 does not reject the null hypothesis.
</div></li><li data-sub-note-item="4-21"><div class="lab-step">Next, to perform t-tests for determining soil composition differences between sites, open a new Excel spreadsheet.
</div></li><li data-sub-note-item="4-22"><div class="lab-step">In cell A1, type 'Site 1:pH', and in cell B1, type 'Site 2:pH'.
</div></li><li data-sub-note-item="4-23"><div class="lab-step">Fill in cells A2 through A6 and cells B2 through B6 with the soil pH collected in the disturbed and undisturbed sites, respectively.
</div></li><li data-sub-note-item="4-24"><div class="lab-step">Click the Data tab in Excel and then select 'Data Analysis'.
</div></li><li data-sub-note-item="4-25"><div class="lab-step">Now scroll down and select 't-Test: Two-Sample Assuming Equal Variances' and select 'Okay'.
</div></li><li data-sub-note-item="4-26"><div class="lab-step">Click the drop down next to Variable 1 Range and highlight cells A2 through A6 in the spreadsheet, then click the dropdown next to Variable 2 Range and highlight cells B2 through B6 in the spreadsheet.
</div></li><li data-sub-note-item="4-27"><div class="lab-step">In the box for hypothesized mean difference, type zero as our null hypothesis. This means that there is no difference between the two disturbance regimes.
</div></li><li data-sub-note-item="4-28"><div class="lab-step">Leave the Labels box unchecked and set the alpha value to 0.05.
</div></li><li data-sub-note-item="4-29"><div class="lab-step">Select 'Output Range', and in the box, click an empty space in the spreadsheet.
</div></li><li data-sub-note-item="4-30"><div class="lab-step">Click 'Okay'. A table of t-Test results should be outputted in the spreadsheet.
</div></li><li data-sub-note-item="4-31"><div class="lab-step">The relevant data are the means for each of the sites and the alpha value. If this second value is less than our alpha value of 0.05, we can reject the null hypothesis that there is no difference in pH between the two sites, and the hypothesis that disturbed versus undisturbed sites differ in their pH value would gain support. If this value is greater than 0.05, however, we may not reject our null hypothesis.
</div></li><li data-sub-note-item="4-32"><div class="lab-step">Repeat this analysis for each of the other three soil variables, creating a new spreadsheet for each of the phosphorus, potassium, and nitrogen elements. 
</div></li><li data-sub-note-item="4-33"><div class="lab-step">Note whether your pH and nutrient level results were different in each condition, and discuss with your group how this might affect the community and diversity in the different sites.
</div></li></ul></li></ol>

Community Diversity: Measuring Biodiversity in Different Areas - Procedure

Topluluk Çeşitliliği

Measuring Biodiversity in Areas with Different Disturbance Regimes
ExpandNOTE: In this experiment, you will compare the biodiversity in a less disturbed ...

Ekoloji

Cellular Respiration

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Quantifying Respiration using Microrespirometers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this experiment, you will measure the rate of cellular respiration for germinating seeds by measuring the rate of exchange for oxygen. As oxygen is consumed to provide energy, germinating seeds release carbon dioxide. This carbon dioxide is absorbed by potassium carbonate and thus the overall gaseous pressure of the respirometer will be reduced. HYPOTHESES: The experimental hypothesis is that germinating seeds will show a greater rate of respiration than control glass beads. Additionally, that at higher temperatures, the rate of cellular respiration in the seeds will increase. The null hypotheses are that both glass beads and germinating seeds will display a similar rate of cellular respiration and that temperature has no effect on respiration.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To set up the control, first remove the plunger from a prepared microrespirometer and label it with a &#8220;CR&#8221; or &#8220;CH&#8221; respectively, depending on whether it is the control room temperature or heated treatment.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Add the glass microbeads to each of the microrespirometers up to the 0.5 mL mark, and push the plunger in the syringe to the 1 mL mark.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place 3 - 4 washers onto the flared end of each microrespirometer to weigh them down while in the water bath.  NOTE: The microrespirometer will sit capillary tube end up.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set the control devices to the side.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To set up the experimental seated respirometers, remove the plunger from a prepared microrespirometer and label it with &#8220;ER&#8221; or &#8220;EH&#8221; respectively, depending on whether it is the experimental room temperature or heated treatment.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Measure 0.5 mL of germinating seeds into the tuberculin syringe of the microrespirometer.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then, push the syringe to the 1 mL mark.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Place 3 - 4 washers onto the flared end of each microrespirometer to weigh them down while in the water bath.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">To use the microrespirometers, carefully press the &#8220;CR&#8221; and &#8220;ER&#8221; microrespirometers into the room temperature water bath. The entire syringe should be submerged, but the capillary tubes should be entirely exposed. Remove water from the bath until this position is achieved. Be sure the top of the capillary tube is open.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Perform the same procedure (step 9) for the &#8220;CH&#8221; and &#8220;EH&#8221; microrespirometers to set up the higher temperature experimental group.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Wait 5 minutes before moving on to allow the temperature in the microrespirometers to equalize with the water bath.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Add a single drop of manometer fluid to the top of each of the four capillary tubes which seals the microrespirometer chambers.  NOTE: If the chambers are working properly, the manometer fluid will be sucked into the capillary chamber.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Use the plunger set in the bath to suck the manometer fluid into the capillary. Stop when the fluid is about halfway down the capillary tube. IMPORTANT: The microrespirometers should not be disturbed once they are added to the bath. Do not bump the table or insert or remove anything to or from the water bath once the chambers have been sealed and equalized.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Mark the bottom edge of the manometer fluid that is closer to the chamber. This represents time point zero.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Set a timer to sound in 5 minutes.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">After 5 minutes, create a new mark where the manometer fluid is on each microrespirometer. Repeat every 5 minutes until 25 minutes has passed or until the manometer fluid has traveled the entire length of the capillary tube.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, remove the respirometers from the water baths.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Measure the distance between each of the marks and record them in Table 1 next to the correct time point. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/8/Table_1_Cellular_respiration.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>  
</div></li><li data-sub-note-item="1-20"><div class="lab-step">During clean up, manometers should either be deconstructed to be reused or disposed of in the trash.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To analyze the data you collected, first plot the data points with the x-axis as time and the y-axis as the distance the manometer moved, representing oxygen consumption.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Calculate the slope of each line using this equation.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Record these values for each microrespirometer in Table 2. NOTE: This value represents the amount of cellular respiration that took place over the course of the experiment.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/8/Table_2_Cellular_respiration.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Plot the respiration rates as a bar graph.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Examine both of the graphs. Consider how your results fit with the hypotheses. Note any observed differences in respiration rate between the control beads and the germinating seeds. Determine if increasing the temperature had any effect on the respiration rate.
</div></li></ul></li></ol>

Cellular Respiration: Quantification using Microrespirometers - Procedure

Hücresel Solunum

Quantifying Respiration using Microrespirometers
ExpandNOTE: In this experiment, you will measure the rate of cellular respiration for germinating seeds ...