<p class="jove_content">현재 프로젝트 개념 펀드의 캔자스의 대학 증거에 의해 투자 되었다. 우리는 cochleae 문화 지원에 대 한 인간의 탯, 그리고 데이비드 조 젠 슨은 취득에 우리 위한 KUMC (캔사스 시, KS)에서 간호 직원을 감사 하 고 싶습니다.</p>

<p class="jove_content">동물 안락사를 포함 하 여 모든 절차는 승인된 기관 동물 관리 및 사용 위원회 (IACUC) 프로토콜 (ACUP #2014-2234) 캔자스의 대학 의료 센터 (KUMC)에 따라 실시 했다.</p>
<p class="jove_content">참고: HWJCs 동의 제공 하는 환자에 의해 기증 된 인간 탯 으로부터 격리 되었고 표본 캔자스의 대학 인간 주제 위원회 (구 IRB #15402)에 의해 승인 하는 프로토콜에 따라 사용 했다.</p>
<p class="jove_title">1. 측 뼈 수확과 달팽이 관 절연</p>
<ol>
	<li>수술가 위, 날카로운 쌍 두개골과 첫 번째 자 궁 경부 척추 사이 절단 하 여 적절 하 게 안락사, 15 주 된 마우스를 목을 벨 다음 스프레이로 소독 (<strong class="xfig">그림 1A</strong>) 70% 에탄올과 머리.</li>
	<li>(<strong class="xfig">그림 1B</strong><strong>-C</strong>, 파선) 수술가 위 같은 날카로운 쌍을 사용 하 여 중간에 화살 평면에서 두개골 이등분</li>
	<li>(<strong class="xfig">그림 1D</strong>, 화살촉) 집게의 쌍을 사용 하 여 두개골에서 뇌 조직을 제거 합니다.</li>
	<li>청각의 존재 및 두개골 및 외관 (<strong class="xfig">1 층 그림</strong>, 화살표)에서 외가도의 내부에 vestibular 신경 뿌리 (<strong class="xfig">그림 1E</strong><strong>-F</strong>, 화살촉) 통해 측 뼈를 식별 합니다. 측 뼈 두개골 (<strong class="xfig">그림 1G</strong>)의 나머지에서 분리 두개골을 통해 조심 스럽게 잘라.<br>참고: 어떤 경우에, 그것 가능 할지도 모른다 섬세 하 게 절단 하지 않고도 측 뼈에서 두개골의 주변 뼈를.</li>
	<li>그래서 팁 달팽이 puncturing 위험 하지 않습니다 약 5 m m (<strong class="xfig">그림 1 H</strong>), 외가도의 개통으로 미세 집게를 삽입 합니다. 부드럽게 깨 물집 (<strong class="xfig">그림 1 H</strong>, 상단 빨간색 음영 처리 된 영역)의 뼈 그래서 그것 골절 (<strong class="xfig">1I 그림</strong>, 빨간 점선 라인).<br>참고: 필요한 경우, 해 부 현미경 수 있습니다 원조이 과정에서 정확 하 게 배치 하 고 악기의 조작.</li>
	<li>미세 집게를 사용 하 여, 놀 리 려 멀리 측 뼈, 뼈 미로 달팽이 관 (<strong class="xfig">그림 1J</strong>, 빨간 괄호)의 노출에서 물집의 나머지.</li>
	<li>페 트리 접시 측 뼈를 충분히 큰 장소 (<em>예를 들어</em>, 30 m m 이상)는 해 부 아래 시야 범위와 함께 충분 한 인산 염 버퍼링 염 분 (PBS) 측 뼈 (<em>예를 들어</em>, 0.5 ~ 1 mL)를 작성.</li>
	<li>향하도록 하는 달팽이 관의 타원형과 둥근 창문이 PBS에 제거 물집과 측 뼈를 잠수함.<br>참고: 달팽이 관에서 vestibular 시스템의 제거 필요는 없습니다,로 vestibular 시스템 역할 잘 조작 하 고 방향을 (<strong class="xfig">1I 그림</strong> 및 <strong class="xfig">그림 2B</strong>) 달팽이 관의 기능에 대 한 핸들.</li>
	<li>집게의 초 미세 쌍을 사용 하는 등골을 통과 stapedial 동맥을 제거 합니다.</li>
	<li>어디 동맥을 통과 하는 등골의 아치를 통해 초 미세 집게의 한 끝을 삽입 하 고 섬세 하 게 위쪽으로 등골을 리프트. Disarticulated 등골 타원형 창에서 리프트 해야 합니다.</li>
	<li>초 미세 집게의 한 팁을 사용 하 여 타원형 펑크 및 windows를 둥근.</li>
	<li>튜빙 28.5 게이지 주사기 가득 1 x PBS를 사용 하 여 (내부 직경 0.28 m m, 외경 0.61 m m) 타원형 창 튜브를 놓고, (<strong class="xfig">그림 2A</strong> <strong> -B</strong>).<br>참고: 튜브의 오프닝 조작할 수 있습니다 정확 하 게 배치 되도록 돕는 초 미세 집게를 사용 하 여.<ol>
		<li>신선한 주사기와 튜브를 사용 하 여 각 달팽이 관에 대 한.</li>
	</ol>
	</li>
	<li>10% 항생제 antimycotic (방지) 및 10% 페니실린-스 (펜-strep) PBS에 5 분 이상 달팽이 관을 통해 제거 하는 perilymph의 2 개 mL perfuse 너무 빨리 너무 많은 압력으로 perfusing는 달팽이 관 내의 미세 구조 손상 것입니다.<br>참고: 수동으로 perfuse 달팽이 관을 통해 액체를 손으로 주사기 운전은 효과적 이다, 관류 펌프 대신으로 사용 될 수 있지만.<ol>
		<li>수동으로 perfusing, 자동 폐쇄 달팽이 관 (<strong class="xfig">그림 2A</strong>) 안정 집게 관류 동안 측 뼈의 vestibular 부분을 파악,의 쌍을 사용 합니다.<br>참고: 필요 하지 않지만 이것은 두 손을 자유롭게 처리 계기; 다른 튜브를 배치할 수 있습니다 하는 동안 한 손으로 주사기 (<strong class="xfig">그림 2A</strong>)를 구동할 수 있다 적절 하 게 (<strong class="xfig">그림 2B</strong>).</li>
		<li>이 단계에서 앞으로, 공기에 불필요 하 게 달팽이 관을 노출 하지 않도록 주의. 유체 순환 차단 유체 공간으로 기포를 도입 하 고 달팽이 관 내의 미세 구조 손상 더 밖으로 건조.</li>
	</ol>
	</li>
	<li>제거 하 고 미세 집게를 사용 하 여 하 모든 나머지 근육 조직과 뼈 파편을 삭제.</li>
	<li>필요한 경우, 최대 7 일간 항생제 솔루션 모든 48 h의 일정 변경에 대 한 10% 방지와 PBS에서 10% 펜 strep 솔루션에 4 ° C에서 고립 된 cochleae를 저장 합니다.</li>
</ol>
<p class="jove_title">2. 인공 처리</p>
<ol>
	<li><strong>Decellularization</strong>
	<ol>
		<li>각 달팽이 관에 대 한 채우기 20 mL 유리 섬광 유리병으로 1% 나트륨 라우릴 황산 드 이온된 (DI) 수, 달팽이 관을 decellularize 하는 데 사용 되는에 (SDS) 솔루션.</li>
		<li>개통은 달팽이 관 통과를 위해 충분히 큰 되도록 면도날을 사용 하 여 플라스틱 7 mL 전송 피 펫의 끝을 잘라.</li>
		<li>전송 피 펫을 사용 하 여, 부드럽게 그릴 달팽이 관 피펫으로에 그래서 그것은 단지 몇 밀리미터에 의해 잘라 가장자리를 전달.</li>
		<li>1 %SDS 솔루션 가득 섬광 유리병에 달팽이 관을 추방.</li>
		<li>전송 피 펫을 사용 하 여 섬광 유리병에 달팽이 관을 통해 드 이온된 (DI) 수에서 1 %SDS 2 개 mL를 순환.</li>
		<li>회전으로 섬광 유리병을 놓고 실내 온도에서 72 h에 대 한 10 rpm 회전 섬광 유리병을 허용 합니다.<ol>
			<li>1 %SDS 솔루션 마다 24 h 72 h 동안 변경 합니다. 결코 공기에 달팽이 관을 노출 하는 SDS를 변경할 때 주의 합니다.</li>
		</ol>
		</li>
		<li>디 물으로 30 분 각각에 대 한 세 번 달팽이 관을 세척.</li></ol></li></ol>SDS 요금; 사용 같은 섬광 유리병에는 세척을 수행 이 단계에서 달팽이 관은 decellularized입니다.
	
	
	<li><strong>Decalcification</strong>
	<ol>
		<li>충분히 빠져들 달팽이 관을 계속 떠나 섬광 유리병에서 나머지 디 물을 제거 합니다.</li>
		<li>Decalcification를 하려면, 달팽이 관을 포함 하는 섬광 유리병에 디 물에 10 %ethylenediaminetetraacetic 산 (EDTA) (0.02 M)의 5 mL을 추가 합니다.</li>
		<li>전송 피 펫을 사용 하 여 섬광 유리병에 달팽이 관을 통해 디 물에 10 %EDTA 2 개 mL를 순환.</li>
		<li>회전에 다시 섬광 유리병을 놓고 실 온에서 72 h에 대 한 10 rpm 회전 섬광 유리병을 허용 합니다. 10 %EDTA 솔루션 마다 24 h 72 h 동안 변경 합니다. 달팽이 관은 공기에 노출 되지 않습니다 있도록 솔루션을 변경할 때 주의 합니다.</li>
		<li>2 h 각 1 x PBS;에 대 한 3 번 달팽이 린스 이 단계에서 달팽이 관은 석.</li>
	</ol>
	</li>
	<li><strong>스토리지</strong>
	<ol>
		<li>10% 방지, 10% 펜 strep 솔루션에 PBS에서 4 ° c.에 최대 72 h에 대 한 cochleae를 저장<br>참고: 긴 저장 기간 항생제 솔루션;의 일정 변경 가능 있을 수 있습니다. 그러나, 처리 된 cochleae의 확장된 저장이이 프로토콜에서 검증 되지 않았습니다.</li>
	</ol>
	</li>

<p class="jove_title">3. 조달 및 hWJCs의 확장</p>
<ol>
	<li>이전에 게시 프로토콜<sup class="xref">22</sup>에 따라 hWJCs를 격리 합니다. 간단히, 3 cm 섹션으로 탯 세그먼트.</li>
	<li>조심 스럽게 만들 메스로 절 개를 얕은 세로 각 탯 선분을 풀 튼의 젤리 탯의 노출에. 집게를 사용 하 여 혈관을 제거.</li>
	<li>조직 잠수함 탯 세그먼트 (<em>예를 들어</em>, 60 mm 페 트리 접시에에서 40 mL) 충분 한 PBS로 두 번 씻는 다. 가늘게 조직 세그먼트 메스를 말하다. 다이제스트 100 mm 페 트리 접시 소화 매체의 50 mL와 조직 (0.2 %2 형 콜라, 1% 페니실린-스, 낮은 혈당에서 Dulbecco의 수정이 글의 중간 (DMEM)).</li>
	<li>소화는 5% CO<sub>2</sub>, 37 ° C 배양 기에서 1 박 50 rpm에서 궤도 통에 중간에 티슈를 놓습니다. 다음 날, 소화 매체 2% 항생제-PBS에서 antimycotic에 1시 16분의 비율로 희석 하 고 실 온에서 500 x g에서 원심 분리 하 여 세포를 작은 (~ 27 ° C) 10 분 삭제 상쾌한에 대 한.</li>
	<li>Resuspend 펠 릿 중간 엽 줄기 세포 성장 매체 (MSCGM)에서 하 고 세포를 재결합. 조직 문화에 7 x 10<sup>3</sup> 셀/c m<sup>2 </sup>의 조밀도에 접시 셀 T-75 플라스 크 취급. MSCGM에서 HWJCs 문화 한 통로 5 실험을 확장 합니다.<br>참고: HWJCs 수 수 하위 양식 큰 T-플라스 크로 (<em>예를 들어</em>, T-150, T-300) 실험에 대 한 hWJCs의 수확량 증가를 뿌리고 때.</li>
</ol>
<p class="jove_title">4입니다. Decellularized Cochleae에 hWJCs의 주입</p>
<ol>
	<li><strong>Decellularized cochleae을 준비</strong>
	<ol>
		<li>무 균 기술을 사용 하 여, 3 시간 30 분 대 한 1 x PBS에 저장된 cochleae 세척.</li>
		<li>Cochleae 24-잘 접시의 별도 우물으로 전송 하 고 37 ° C의 1 mL를 추가 MSCGM를 미리 예 열.</li>
		<li>Cochleae는 5% CO<sub>2</sub>, 세포의 주입 하기 전에 1 h의 최소 37 ° C 세포 문화 인큐베이터에서에서 품 어.</li>
	</ol>
	</li>
	<li><strong>해리 hWJCs 및 resuspend</strong>
	<ol>
		<li>37 ° c 워시 hWJCs 전 두 번 1 x PBS를 따뜻하게합니다.</li>
		<li>37 ° c 충분 한 사전 예 열 셀 선박 표면에 0.5 %EDTA 트립 신.</li>
		<li>HWJCs는 5% CO<sub>2</sub>, 37 ° C 세포 문화 인큐베이터에서에서 최대 5 분을 품 어.<ol>
			<li>세포의 90%는 거꾸로 현미경 세포를 확인 하 여 세포 문화 표면에서 분리 된 확인 합니다.<br>참고: 때 문화 배 도청 부드럽게 이동 하는 셀 분리 했습니다. 셀 고정, 남아 있는 분리 하지 있다.</li>
			<li>부드럽게 연결 된 셀을 더 꺼내 려 플라스 크의 측면을 누릅니다.</li>
		</ol>
		</li>
		<li>무 균 기술을 사용 하 여, 해당 볼륨 MSCGM의 Trypsin의 볼륨을 포함 하는 50 mL 원뿔 튜브에 문화 선박에서 전송 hWJCs는 세포 분열을 사용.</li>
		<li>실 온에서 500 x g에서 centrifuging 여는 hWJCs 작은 (~ 27 ° C) 5 분.</li>
		<li>상쾌한, 발음 하 고 500, 000 셀/mL의 농도에서 MSCGM에 hWJCs를 resuspend.</li>
	</ol>
	</li>
	<li><strong>Cochleae perfuse</strong>
	<ol>
		<li>Cochleae 전송 피 펫을 사용 하 여 새로운 24-잘 접시에 전송 합니다.</li>
		<li>어떤 달팽이 관 밖으로 건조 하지 않도록 하려면 각 달팽이 관에 MSCGM의 한 방울을 추가 합니다.</li>
		<li>섬세 하 게 타원형과 둥근 창문을 향하도록 하 초 미세 집게를 사용 하 여 달팽이 관을 방향을 정하십시오.<br>참고: 주의 극단적인 달팽이 관 손상 쉽게 각 달팽이 관을 직접 처리 하는 경우.</li>
		<li>살 균 28.5 게이지 인슐린 주사기를 사용 하 여 연결 된 튜빙, 0.2 mL resuspended hWJCs (100000 셀)를 그립니다.</li>
		<li>멸 균된 미세 집게를 사용 하 여 케이블의 튜빙을 타원형 창에 놓으십시오.</li>
		<li>섬세 하 게 그리고 천천히 perfuse 폴 리 에틸렌 튜브에 연결 28.5 게이지 인슐린 주사기를 사용 하 여 resuspended hWJCs의 0.2 mL와 달팽이 관 (외경: 0.61 m m, 내경: 0.28 m m) 약 5 분 이상.</li>
		<li>관류, 후 37 ° C의 0.8 mL를 추가할 필요는 잘 포함 하는 잘에서 1 mL 전체 볼륨을가지고 달팽이 관에 MSCGM를 미리 따뜻하게.</li>
		<li>각 달팽이 관, 신선한 주사기를 사용 하 고 각 시간을 튜브에 대 한 4.3.3-4.3.7 단계를 반복 합니다.</li>
		<li>5% CO<sub>2</sub>, 37 ° C 세포 문화 인큐베이터, 끼얹는다 cochleae를 놓고 미디어 주 당 3 시간을 변경 합니다.</li>
	</ol>
	</li>
</ol>
<p class="jove_title">5. 달팽이 수확 및 보존</p>
<p class="jove_content">참고: Cochleae 그리고 경작 수 있습니다 최대 30 일 후 관류 시간 언제 든 지 수확.</p>
<ol>
	<li>Cochleae를 유지 하려면 1 x PBS 락 플랫폼에 4 ° C에서 하룻밤에 4 %paraformaldehyde 수정 했다.</li>
	<li>고정, 후 5 분에 대 한 세 번 1 x PBS 가진 cochleae 세척.</li>
	<li>점차적으로 에탄올과 달팽이 관을 탈수 하 고 파라핀에 포함 하기 전에 지방 족 탄화수소 용 매로 취소 합니다.</li>
	<li>에 톰 및 마운트를 사용 하 여 10 µ m의 두께로 섹션 샘플 현미경 슬라이드 유리.<br>참고: 샘플 준비가 이제 조직학 immunohistochemical 처리 표준 프로토콜을 사용 하 여 또는.</li>
</ol>

<ol>
	<li>Raphael, Y., Altschuler, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+innervation+of+the+cochlea.">Structure and innervation of the cochlea.</a> <em style='font-style: italic'>Brain Res Bull</em>. <b>60</b> (5-6), 397-422 (2003).</li><li>LeMasurier, M., Gillespie, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair-Cell+Mechanotransduction+and+Cochlear+Amplification.">Hair-Cell Mechanotransduction and Cochlear Amplification.</a> <em style='font-style: italic'>Neuron</em>. <b>48</b> (3), 403-415 (2005).</li><li>Neal, 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=Hair+cell+counts+in+a+rat+model+of+sound+damage:+Effects+of+tissue+preparation+&amp;+identification+of+regions+of+hair+cell+loss.">Hair cell counts in a rat model of sound damage: Effects of tissue preparation &amp; identification of regions of hair cell loss.</a> <em style='font-style: italic'>Hear Res</em>. <b>328</b>, 120-132 (2015).</li><li>Ivory, R., Kane, R., Diaz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+hearing+loss:+a+recreational+noise+perspective.">Noise-induced hearing loss: a recreational noise perspective.</a> <em style='font-style: italic'>Curr Opin Otolaryngol Head Neck Surg</em>. <b>22</b> (5), 394-398 (2014).</li><li>Stucken, E. Z., Hong, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+hearing+loss:+an+occupational+medicine+perspective.">Noise-induced hearing loss: an occupational medicine perspective.</a> <em style='font-style: italic'>Curr Opin Otolaryngol Head Neck Surg</em>. <b>22</b> (5), 388-393 (2014).</li><li>Dille, M. F., 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=Tinnitus+onset+rates+from+chemotherapeutic+agents+and+ototoxic+antibiotics:+results+of+a+large+prospective+study.">Tinnitus onset rates from chemotherapeutic agents and ototoxic antibiotics: results of a large prospective study.</a> <em style='font-style: italic'>J Am Acad Audiol</em>. <b>21</b> (6), 409-417 (2010).</li><li>Sajjadi, H., Paparella, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meniere's+disease.">Meniere's disease.</a> <em style='font-style: italic'>Lancet</em>. <b>372</b> (9636), 406-414 (2008).</li><li>House, J. W., Brackmann, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinnitus:+surgical+treatment.">Tinnitus: surgical treatment.</a> <em style='font-style: italic'>Ciba Found Symp</em>. <b>85</b>, 204-216 (1981).</li><li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+age-related+hearing+loss+by+early+noise+exposure:+evidence+of+a+misspent+youth.">Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth.</a> <em style='font-style: italic'>J Neurosci</em>. <b>26</b> (7), 2115-2123 (2006).</li><li>Ryals, B. M., 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=Avian+species+differences+in+susceptibility+to+noise+exposure.">Avian species differences in susceptibility to noise exposure.</a> <em style='font-style: italic'>Hear Res</em>. <b>131</b> (1-2), 71-88 (1999).</li><li>Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D., Hashino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+inner+ear+sensory+epithelia+from+pluripotent+stem+cells+in+3D+culture.">Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture.</a> <em style='font-style: italic'>Nature</em>. <b>500</b> (7461), 217-221 (2013).</li><li>Sekiya, T., 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=Cell+transplantation+to+the+auditory+nerve+and+cochlear+duct.">Cell transplantation to the auditory nerve and cochlear duct.</a> <em style='font-style: italic'>Exp Neurol</em>. <b>198</b> (1), 12-24 (2006).</li><li>Shi, F., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+for+replacement+of+auditory+neurons+by+stem+cells.">Prospects for replacement of auditory neurons by stem cells.</a> <em style='font-style: italic'>Hear Res</em>. <b>297</b>, 106-112 (2013).</li><li>Mellott, A. J., Shinogle, H. E., Nelson-Brantley, J. G., Detamore, M. S., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+decellularized+cochleae+as+scaffolds+for+inner+ear+tissue+engineering.">Exploiting decellularized cochleae as scaffolds for inner ear tissue engineering.</a> <em style='font-style: italic'>Stem Cell Res Ther</em>. <b>8</b>, (2017).</li><li>Santi, P. A., Johnson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+ear+tissues+as+scaffolds+for+stem+cell+differentiation.">Decellularized ear tissues as scaffolds for stem cell differentiation.</a> <em style='font-style: italic'>J Assoc Res Otolaryngol</em>. <b>14</b> (1), 3-15 (2013).</li><li>Davies, D., Holley, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+alpha+3+and+alpha+6+integrins+in+the+developing+mouse+inner+ear.">Differential expression of alpha 3 and alpha 6 integrins in the developing mouse inner ear.</a> <em style='font-style: italic'>J Comp Neurol</em>. <b>445</b> (2), 122-132 (2002).</li><li>Gerchman, E., Hilfer, S. R., Brown, J. 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">우리는 성공적으로 복잡 한 3 차원 조직 발판으로 달팽이 관의 사용을 허용 하는 decellularization 과정을 통해 달팽이 관에서 네이티브 인공 세포를 제거할 수 있습니다 시연 했다. 산 티 <em>외</em>. <sup class="xref">15</sup> decellularizing cochleae에 대 한 초기 방법을 개발 하 고 정확 하 게 빛 시트 현미경<sup class="xref">23</sup>의 도움으로 많은 인공 구조를 통해 볼륨 견적 했다. 이러한 초기 작업 조직 엔지니어링과 세포 문화 기술을 여기에 대 한 강력한 기반으로 재직 했습니다. Decellularized 달팽이 관 수 성공적으로 융합 세포, 그리고 그 시간의 연장된 기간에 대 한 교양. 다양 한 동물에서에서 cochleae 수 이론적으로 세포 유형의 다양 한 유사 하 게와 함께 활용할 수 있습니다. 이러한 조합을 허용 여기에 제시 된 수많은 가능한 실험적인 디자인, 감각 상피 개발<sup class="xref">14</sup>검사, 약물의 새로운 제약 에이전트<sup class="xref"> 테스트 하는 응용 프로그램 엔지니어링 조직에 활용 하는 기법 24</sup>, 와우 복구의 개발 모델<sup class="xref">25</sup>. 그러나, 이러한 기술의 광범위 한 적용에도 불구 하 고 그들은 되지 않습니다 제한 없이.</p>
<p class="jove_content">현재 기술의 한계 중 하나는 관류 개인에 따라 변수입니다. 달팽이 관에 대 한 홀더를 개발 하 고 주사기 펌프를 사용 하 여 perfusions를 수행 과정의 성공과 정확성 증가할 수 있습니다. 또한, 결정적으로 증명 하는 성공적인 decellularization 잠재적으로 여러 가지 방법을 통해 수행할 수 있는. 우리 DAPI 얼룩이 남아 있는 세포 핵을 계량 하는 데 사용 된 고용 하 고 우리가 관찰 아니 잔여 핵 게시물-decellularization. 또한, 표준 DNA 정량화 분석 실험 시 약, 녹색 염료 picogram 양의 DNA 핵 산 나머지, 시각적으로 핵의 작은 파편의 존재를 식별 DAPI 보다 더 민감한 수 있는 식별을 검색 활용 decellularized 조직<sup class="xref">26</sup>에 있는 산. 어떤 기법은 성공적인 decellularization의 유효성을 검사 하는 데 사용 됩니다에 발판으로 활용 하 여 이전 개별 달팽이 관의 완전 한 decellularization를 하지 않을 수 있습니다. 그럼에도 불구 하 고, 우리는 성공적이 고 강력한 decellularization 과정을 발견 했다.</p>
<p class="jove_content">현재 프로토콜에서 두 개의 가장 중요 한 단계는 고립 그리고 cochleae의 처리는 cochleae의 관류. 달팽이 관은 섬세 하 고 취 성 달팽이 관 처음 격리 된 때 신중을 기울여야 합니다. 집게와 달팽이 관을 처리 하는 동안 너무 많은 힘을 적용 하는 경우는 달팽이 관 수 조각화 됩니다. 따라서, decellularization 및 decalcification 전에 각 달팽이 관을 부드럽게 처리 하는 것이 필수적 이다. Vestibular 시스템 그대로, 겸 자와 함께 복잡 한 내 귀를 잡아와 달팽이 관을 향하는 큰 핸들로 사용 됩니다. 마찬가지로, perfusing 세포와 달팽이 관, 뒤에 관류 압력 너무 중대 해서는 안, 그렇지 않으면 세포는 관류 살아날 수 있습니다. 현재 프로토콜에서 설명 했 듯이, 1 mL 인슐린 주사기의 끝에 튜브를 부착 한 손으로, 및 perfusing 다른 손으로 달팽이 관을 통해 셀에 정밀도와 달팽이 관의 쉽게 처리 할 수 있습니다.</p>
<p class="jove_content">다음 단계로 그것 자연 인공 ECM은 줄기 세포의 분화를 유도 하 고 있다와 그래서, 달팽이 관에서 특정 ECM 구성 요소를 셀에 변경 내용을 유도 확인을 발달 및 기능 표시를 평가 하는 것입니다. 다른 종류의 세포 (<em>예를들면</em>, 줄기 세포, neuroprogenitors, 섬유 아 세포, <em>등</em>) 모니터 세포 분화 및 행동에 달팽이 관을 통해 perfusing 다른 매혹적인 후속 조사 될 것 이다. 또한, 세포 유전자 변형 또는 성장 인자 차별화 프로토콜에 노출 되는 인공 ECM neurosensory 개발을 지원 하는 방법에 대 한 새로운 통찰력을 제공 하 고 청문회에서 함수 어쩌면 수 있습니다. 따라서, 현재 프로토콜 인공 ECM를 사용 하 여 내가 neurosensory 개발 및 유지 보수, 5 월 1 일 새로운 치료법의 개발로 이어질 청력 손실 복원 연구에 중요 한 첫 단계입니다.</p>

<p class="jove_content">달팽이 관은 측 뼈에서 발견 하는 복잡 한 나선형 구조. 그것은 외부 뼈 미로 동심, 내부 membranous 미로<sup class="xref">1</sup>로 구성. Membranous 미로 3 유체 공간 구성: 스칼라 vestibuli, 스칼라 언론과 Scala tympani<sup class="xref">1</sup>. 스칼라 미디어 하우스 수많은 종류의 세포로 구성 되어, 감각 상피, 하지만 감각 머리 세포 (HC), 신경 충 동<sup class="xref">2</sup>음파 기계적 에너지 transduce는 특정 관심의. 음향 외상<sup class="xref">3</sup><sup>,</sup><sup>,</sup><sup class="xref">4</sup><sup class="xref">5</sup>, 약<sup class="xref">6</sup>, 질병<sup class="xref">7</sup><sup>,</sup><sup class="xref">8</sup>, 및 노화<sup class="xref">9</sup> 노출 모두 HC 죽음 통해 장애인된 청각 기능 발생할 수 있습니다. 포유류에서 머리 세포 손실 부상<sup class="xref">10</sup>후 재생성 수 조류 HCs 달리 영구적 이다.</p>
<p class="jove_content">다양 한 현대 연구 노력 하고자 했다 손실된 HCs 복원 특정 실험적인 접근 다르지만. 감각 상피에 유전자 발현의 조작 및 몸 밖에 서 분화 하는 줄기 세포의 주입은 필드에서 지배적인 접근 인공 organoids로 줄기 세포를 분화 하는 유도 방법 있다 지만 시도<sup class="xref">11</sup><sup>,</sup><sup>,</sup><sup class="xref">12</sup><sup class="xref">13</sup>. 이 방법의 각각은 줄기 세포 나 줄기 세포; 사용 발달 큐에 직접 의존 그러나, 두 번째 공유 하 고 잠재적으로 중요 한 요소는 달팽이 관 자체의 ECM<sup class="xref">14</sup><sup>,</sup><sup class="xref">15</sup>.</p>
<p class="jove_content">ECM 뿐만 아니라 세포와 조직, 세포 접착, 확산, 생존, 및 마이그레이션에 대 한 서피스를 포함 하지만 또한 HCs와 나선형 ganglion<sup class="xref">15</sup><sup>,</sup><sup class="xref">16의 개발에 중요 한 역할에 대 한 물리적 지원 제공 </sup> <sup>,</sup><sup class="xref">17</sup>. 자연스럽 게 발생 ECM는 세포 표현 형 결정 및 세포 접착, 확산, 그리고 생존<sup class="xref">18</sup>가이드 수 유도 신호를 제공 합니다. 따라서, 교양된 hWJCs와 함께에서 decellularized 달팽이 관의 사용 ECM HC 재생의 역할을 탐구 하는 독특한 기회를 제공 합니다. HWJCs는 중간 엽 줄기 세포<sup class="xref">19</sup>처럼 인간의 탯에서 고립 된 쉽게 사용할 수, 비 논란이 셀 유형입니다. HWJCs는 neurosensory 세포 계보<sup class="xref">20</sup><sup>,</sup><sup class="xref">21</sup>아래로 차별 하는 능력을 보여주었다. 따라서, 현재 프로토콜 절연, decellularization, 및 cochleae 내가 조직 공학에 대 한 hWJCs와 C57BL 마우스 시체에서의 관류 자세히 설명합니다.</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">포유류에서 청각 부족을 재생성 하는 기능을 촉진 하는 mechanosensory 세포는 청력 손실에 대 한 치료를 제한 했다. 현재 재생 의학 전략 이식 줄기 세포 또는 주변 청각 상실을 해결 하기 위해 손상 된 줄기 세포의 교체를 장려 하는 내가 지원 세포의 유전자 조작에 집중 했다. 그러나, 기질 (ECM) 유도 하 고 세포의 기능 유지에 중요 한 역할을 재생할 수 있습니다 그리고 잘 조사 하지. 인공 와우를 사용 하 여 성체 줄기 세포를 성장 하는 발판으로 ECM 어떻게 구성 및 extracellular 환경 아키텍처 셀에 에이즈 청력 기능을 유지 하는 독특한 통찰력을 제공할 수 있습니다. 여기 우리는 분리 하 고 건설 기계 성인 줄기 세포 끼얹는다 수락으로 사용 하는 마우스에서 cochleae를 decellularizing 하는 방법을 제시. 현재 프로토콜에서 cochleae 안락사 쥐, decellularized, 그리고 석 으로부터 격리 됩니다. 그 후, 탯 줄에서 고립 된 인간의 튼 젤리 셀 (hWJCs) 각 달팽이 관에 끼얹는다 신중 하 게 했다. cochleae 생물 반응 기, 사용 하 고 셀 분석에 대 한 처리를 진행 하기 전에 30 일 동안 배양 되었다. Decellularized cochleae 식별 extracellular 구조를 유지 하지만 DNA의 현저한 조각 또는 셀의 존재를 공개 하지 않았다. 셀 달팽이 관에 끼얹는다 인테리어와 달팽이 관의 외관의 대부분을 침공 하 고 30 일의 기간 동안 사고 없이 성장 했다. 따라서, 현재 방법 어떻게 인공 ECM에 영향을 미치는 세포 발달 및 행동 연구를 사용할 수 있습니다.</p>

<p class="jove_content" fo:keep-together.within-page="1">여기에 제시 하는 방법을 사용 하 여, cochleae의 성공적인 decellularization 4'를 통해 DNA의 유무를 검사 하 여 평가 했다 6-diamidino-2-phenylindole (DAPI) 얼룩. Cochleae는 DNA 이었다 decellularized 달팽이 관 내에서 확인 되지 않는 경우 완전히 decellularized로 간주 됐다. Decellularization 또는 decalcification을 받을 하지 않았다 이전 실험에서 네이티브 달팽이 관 구조와 전통적으로 C57BL 마우스 달팽이 관에 있는 세포를 설명 하기 위해 긍정적인 제어로 사용 되었다. 어떤 hWJCs를 주입 하지 않은, decellularized-석된 달팽이 관, 부정적인 컨트롤으로 사용 되었다. 아니 정량 DAPI 스테인드 세포 핵 조직 단면도 (<strong class="xfig">그림 3</strong>)의 어떤 지역에서 관찰 되었다. 두 개의 cochleae decellularized 고 석, 되었고 현재 프로젝트에 대 한 hWJCs 주입. HWJCs와 함께 끼얹는다 했다 decellularized-석된 cochleae 수많은 DAPI를 달팽이와 달팽이 관 (<strong>표 1</strong>) 외부 뼈 껍질에 성장 핵 스테인드 관찰 되었다. 총 예상 셀 셀 밀도 생긴 첫 번째 달팽이 관 후 30 일 문화 (<strong class="xfig">그림 4</strong> 및 <strong class="xfig">그림 5</strong>)와 두 번째 달팽이 관에 대 한 문화의 30 일 후 118,732 세포/달팽이 관 113,759 세포/달팽이 관을 했다 (<strong class="xfig">그림 6</strong> 과 <strong class="xfig">그림 7</strong>)입니다. 이러한 셀 밀도 추정 Scala tympani, 스칼라, 그리고 스칼라 현관, 언론과 구조 3 유체 공간 내에서 볼륨을 포함 하지만 이러한 견적 제외 나머지 골 질 미로. 또한, 각 달팽이 관에서 샘플 Hematoxylin와 오신 스테인드 했다 (H &#38; E) 세포와 각 달팽이 관 (<strong class="xfig">그림 8</strong>) 내에서 해 부 특징의 존재를 보여. 와우 마이크로 구조의 심한 해부학 모든 스테인드 섹션;에 걸쳐 주로 불변에 남아 있었다 그러나, 셀 (<strong class="xfig">그림 8B</strong>) decellularized-석된 섹션에서 식별 했다. 세포 핵의 식별 네이티브 달팽이 관 (<strong class="xfig">그림 8A</strong>)과 셀 (<strong class="xfig">그림 8C</strong><strong>-</strong><strong>D</strong>) 주입 decellularized 석된 cochleae 사이 극적으로 달랐다.</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>그림 1: 측 뼈 절연 마우스.</strong> 일단 마우스 적절 하 게 안락사 되었습니다, 두개골의 기지와 첫 번째 자 궁 경부 척추 사이 절단 하 여 동물을 목을 벨 고 에탄올 소독 (<strong>A</strong>) 머리를 스프레이. 수술가 위, 뇌 조직 및 뼈를 통해 절단 날카로운 쌍을 사용 하 여 섹션<strong>B</strong><strong>-</strong>(<strong>C</strong>)의 화살 평면에서 두개골 이등분 Bisected 마우스 머리 (<strong>D</strong>, 없는 실험적인 작품에 대 한 제거 하는 낮은 mandible) 뇌 조직 측 뼈를 제거 해야 합니다 다음. 청각과 vestibular 신경 통과 (<strong>E</strong>, 빨간 화살촉)는 유용한 두 foramina 측 뼈를 성공적으로 식별에 대 한 단서. 두개골 (<strong>F</strong>)에서 육체를 제거 합니다. 외부 귀 운하 (<strong>F</strong>, 화살표, 맨 위 패널) 뿐만 아니라 유용한 외부 큐를 제공합니다. 신중 하 게 멀리 측 고립 된 뼈 (<strong>G</strong>)에 그냥 떠나 초과 뼈 손질. 물집 (<strong>H</strong>, 상위 패널, 붉은 강조 표시 된 영역) 제거 해야 합니다 다음 수 섬세 하 게 잘 집게 (<strong>H</strong>, 낮은 패널)을 사용 하 여. 물집 얇은 뼈 이며, 수 있다 쉽게 부서진 떨어져 (<strong>나</strong>) 뼈 미로 달팽이 관의 계시 (<strong>J</strong>, 빨간 괄호) 될 때까지. 모든 이미지에 스케일 바는 1 m m. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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>그림 2: 인공 관류.</strong> 측 뼈 고립 되어가 물집 제거 되었습니다 일단 달팽이 관 다음 설정할 수 있습니다 현미경에 관류에 대 한. 튜빙 수동으로 구동된 주사기를 사용 하 여 perfusing (<strong>A</strong>, 라인을 점선) 때 자체 닫는 집게의 쌍을 사용 하 여 달팽이 관을 안정화 하는 데 도움이 수 있습니다. 부드럽게 측 뼈 (<strong>B</strong>)의 vestibular 부분에는 집게를 연결 하 고 페 트리 접시 (<strong>A</strong>)의 입술에 대 한 겸 휴식. 이것은 두 손을 자유롭게 관류 동안 악기를 조작 하. 한 손으로 다음, 액체의 유량을 제어 하는 동안 다른 손 집게 (<strong>B</strong>)의 두 번째 쌍을 사용 하 여 타원형 창을 통해 튜브를 배치할 수 있습니다 주사기를 조작할 수 있습니다. 튜브의 무료 끝 각도 절단 허용 쉽게 위치, 튜브의 시야를 차단 하지 않고 올바로 배치를 허용 합니다. 눈금 막대는 1 m m. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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>그림 3: decellularized 마우스 달팽이 관의 조직 단면도.</strong> 똑바로 피 형광 현미경 (10 X 접 안 렌즈 및 20 X 목표) 200 X의 확대에서 샘플 이미지를 사용 했습니다. 이미지는 약 4 × 5 몽타주를 만들려고 함께 바느질 했다. 셀의 표시 부재 얼룩 핵 DAPI 패널 <strong>A </strong>DAPI 공통 필터 집합 (350 nm 여기, 470 nm 방출), 그리고 설쳐를 사용 하 여 fluorescein isothiocyanate (FITC) 일반적인 필터를 사용 하 여 autofluorescence의 회색 음영 이미지에에서 표시 됩니다. 세트 (490 nm 여기, 525 nm 방출), 해부학 적 랜드마크를 제공 하는 패널 <strong>B</strong>에 표시 됩니다. 자동된 셀 수 <strong>A</strong> 와 <strong>B</strong>두 패널에 delineated 관심의 3 개 지역에서 수행 했다. 총 세포 수는 전체 조직 단면도 (<strong>1</strong>, 흰색 실선)과 달팽이 관 (<strong>2</strong>, 파선)에 대 한 계산 했다. 이 두 숫자 달팽이 관 이상으로 하지만 조직 섹션 (<strong>3</strong>, 점선 및 파선 사이의 공간)의 외부, 뼈 가장자리에서 뼈 구조에 있는 셀의 수를 계산 하는 데 사용 되었습니다. 모든 이미지에 규모 바 500 µ m입니다.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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>그림 4: decellularized 마우스 달팽이 관 1의 가까이 modiolar 섹션 들어갈 hWJCs.</strong> DAPI 핵 얼룩 <strong>A</strong> 해부학 적 랜드마크를 제공 하는 녹색 채널에서 설쳐, 회색조 autofluorescence에 중첩 패널에 표시 됩니다. 자동 셀 카운트 패널 <strong>A</strong>에 delineated 관심의 3 개 지역에서 수행 되었다. 총 세포 수는 전체 조직 단면도 (<strong>1</strong>, 흰색 실선)과 달팽이 관 (<strong>2</strong>, 파선)에 대 한 계산 했다. 이 두 숫자 달팽이 관 이상으로 하지만 조직 섹션 (<strong>3</strong>, 점선 및 파선 사이의 공간)의 외부, 뼈 가장자리에서 뼈 구조에 있는 셀의 수를 계산 하는 데 사용 되었습니다. <strong>B</strong>, 패널에 표시 됩니다 절연 DAPI 얼룩이 지 고 자동화 된 정량화 사용 DAPI 얼룩의 임계값 <strong>C</strong>패널에 표시 됩니다. 모든 이미지에 스케일 바는 500 µ m. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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>그림 5: decellularized 마우스 달팽이 관 1의 가까이 modiolar 섹션의 높은 확대 보기 들어갈 hWJCs.</strong> DAPI 핵 얼룩 <strong>A</strong> 해부학 적 랜드마크를 제공 하는 녹색 채널에서 autofluorescence의 설쳐, 회색 음영 이미지에 중첩 패널에 표시 됩니다. <strong>B</strong>, 패널에 표시 됩니다 절연 DAPI 얼룩이 지 고 자동된 셀 카운팅 하는 동안 사용 되는 임계값 이미지 <strong>C</strong>패널에 표시 됩니다. 유체 공간 및 달팽이 관 내에 위치 해 미세한 마이크로 구조는 decellularization 동안 잘 보존 실험의 문화 단계 세포. 스칼라 Vestibuli (SV), Scala Tympani (SM), Basilar 막 (BM), Tectorial Membrane (TM), 나선 Limbus (L), 로젠 탈의 운하 (RC), Modiolus (M), 나선형 인 대 (SL), 그리고 흔 Vascularis (<strong>*</strong>). Reissner의 막 조직 섹션에 명확 하 게 정의 되지 않았다 그리고 결과적으로 스칼라 미디어 명확 하 게 정의 되지 않았습니다. 스케일 바의 모든 이미지에는 250 µ m. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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>그림 6: modiolar에 가까운 부분의 hWJCs 주입 했다 decellularized 마우스 달팽이 2.</strong> DAPI 핵 얼룩 패널 <strong>A</strong>, 해부학 적 랜드마크를 제공 하는 녹색 채널에서 회색조 autofluorescence에 중첩에 표시 됩니다. 자동 셀 카운트 패널 <strong>A</strong>에 delineated 관심의 3 개 지역에서 수행 되었다. 총 세포 수는 전체 조직 단면도 (<strong>1</strong>, 흰색 실선)과 달팽이 관 (<strong>2</strong>, 파선)에 대 한 계산 했다. 이 두 숫자 달팽이 관 이상으로 하지만 조직 섹션 (<strong>3</strong>, 점선 및 파선 사이의 공간)의 외부, 뼈 가장자리에서 뼈 구조에 있는 셀의 수를 계산 하는 데 사용 되었습니다. <strong>B</strong>, 패널에 표시 됩니다 절연 DAPI 얼룩이 지 고 자동화 된 정량화 사용 DAPI 얼룩의 임계값 <strong>C</strong>패널에 표시 됩니다. 모든 이미지에 스케일 바는 500 µ m. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig6large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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>그림 7: hWJCs 주입 했다 decellularized 마우스 달팽이 2의 가까이 modiolar 섹션의 높은 확대 보기.</strong> DAPI 핵 얼룩 패널 <strong>A</strong>, 해부학 적 랜드마크를 제공 하는 녹색 채널에서 회색조 autofluorescence에 중첩에 표시 됩니다. <strong>B</strong>, 패널에 표시 됩니다 절연 DAPI 얼룩이 지 고 자동된 셀 카운팅 하는 동안 사용 되는 임계값 이미지 <strong>C</strong>패널에 표시 됩니다. 유체 공간 및 미세한 마이크로 구조는 달팽이 관 내에 위치 해 실험의 decellularization 및 셀 문화 단계 동안 유지 됩니다. 스칼라 Vestibuli (SV), Scala Tympani (SM), Basilar 막 (BM), Tectorial Membrane (TM), 나선 Limbus (L), 로젠 탈의 운하 (RC), Modiolus (M), 나선형 인 대 (SL), 그리고 흔 Vascularis (<strong>*</strong>). Reissner의 막 조직 섹션에 명확 하 게 정의 되지 않았다 그리고 결과적으로 스칼라 미디어 명확 하 게 정의 되지 않았습니다. 스케일 바의 모든 이미지에는 250 µ m. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig7large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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>그림 8: Hematoxylin와 오신 얼룩 제어 및 치료 cochleae.</strong> 되며 및 네이티브 셀 현재 일반적인 달팽이 관을 표시 하는 섹션을 스테인드 오신 패널 <strong>A</strong>에 표시 됩니다. 패널 <strong>B</strong> 패널 <strong>A</strong>,으로 동일한 구조를 포함 하지만 세포 외 매트릭스 뒤에 완전히 decellularized 달팽이 관에서. 2 개의 이전 표시 cochleae hWJCs 주입 패널 <strong>C</strong> 와 <strong>D</strong>에서 볼 수 있습니다. 비록 특정 세포 인구와 위치는 극적으로 변경 된 총 인공 해부학 주로 decellularization 및 세포 배양 프로세스를 통해 그대로 남아 있습니다. 스칼라 Vestibuli (SV), Scala Tympani (SM), Basilar 막 (BM), Tectorial Membrane (TM), 나선 Limbus (L), 로젠 탈의 운하 (RC), Modiolus (M), 나선형 인 대 (SL), 그리고 흔 Vascularis (*). 스케일 바의 모든 이미지에는 250 µ m. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56523/56523fig8large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a></p>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td><strong>A 1 달팽이 관 + 셀</strong></td>
			<td><strong>A2 달팽이 관 + 셀</strong></td>
			<td><strong>B2 부정적</strong></td></tr></tbody></table>제어 달팽이 관
		
		<tr>
			<td><strong>외부 골 질 미로</strong></td>
			<td>3,758</td>
			<td>549</td>
			<td>0</td>
		</tr>
		<tr>
			<td><strong>뼈가 외부 달팽이 관 공간</strong></td>
			<td>248</td>
			<td>586</td>
			<td>0</td>
		</tr>
		<tr>
			<td><strong>달팽이 관 내</strong></td>
			<td>518</td>
			<td>701</td>
			<td>0</td>
		</tr>
		<tr>
			<td><strong>총 셀</strong></td>
			<td>4,524</td>
			<td>1,836</td>
			<td>0</td>
		</tr>
		<tr>
			<td><strong>섹션 볼륨 (μ)</strong></td>
			<td>0.0077</td>
			<td>0.0100</td>
			<td>0.0147</td>
		</tr>
		<tr>
			<td><strong>달팽이 관의 볼륨의 %</strong></td>
			<td>0.46%</td>
			<td>0.59%</td>
			<td>0.87%</td>
		</tr>
		<tr>
			<td><strong>달팽이 관 내의 셀 밀도 추정</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>표 1: 셀 수.</strong> DAPI 스테인드 인공 섹션 thresholded 되었고 관심 (밖에 서 뼈 미 궁, 측 뼈 인공 액체 공간 및 달팽이 관 내) 3 겹치지 않는 영역에 ImageJ의 자동 계산 도구를 사용 하 여 정량. ImageJ, 달팽이 관 단면적 측정 또한 되었다 그리고이 값 섹션 두께 (10 µ m)와 조합에서 섹션 볼륨을 계산 하는 데 사용 되었다. 산 티 <em>외</em> 에 의해 총 인공 볼륨의 견적을 사용 하 여 <sup class="xref">23</sup>, 주어진된 섹션의 상대 인공 볼륨 계산 했다. 전체 달팽이 관의 셀 밀도 짝된 셀 수 조합에 지정 된 섹션의 상대 인공 볼륨을 사용 하 여 추정 되었다.</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

내가 조직 공학에 대 한 마우스 Cochleae Decellularizing에 대 한 프로토콜

<p class="jove_content">이 프로토콜의 목표 decellularize 알려줍니다 마우스 cochleae 조직 공학 응용 프로그램에 대 한 공중 발판으로 이용 하는 효과적인 방법 설명 하는 것입니다.</p>

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

a-protocol-for-decellularizing-mouse-cochleae-for-inner-ear-tissue

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.7K 조회수.  University of Kansas Medical Center. 이 프로토콜의 목표 decellularize 알려줍니다 마우스 cochleae 조직 공학 응용 프로그램에 대 한 공중 발판으로 이용 하는 효과적인 방법 설명 하는 것입니다.

비디오: 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

진화적 관계

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

교육

JoVE Lab Manual

생물학

진화

Extraction

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

추출

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

	Cellulose RecoveryExpand
	Extraction and filtration can ...

화학

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

전해 전지

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

갈바닉 셀

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

세포 분열

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 ...

세포 과정

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

세포 구조

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

기초

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

유기체의 유전학

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 ...

유전학

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

미생물 및 곰팡이 다양성

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

커뮤니티 다양성

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

생태학

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

세포 호흡

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