Yazar Bhagawat Subramanian, Yi (Jason) Chang, Paul Randazzo ve Carole üst okuma ve el yazması üzerinde yorum için teşekkür etmek istiyor. Bu eser Ulusal Kanser Enstitüsü Intramural araştırma programı tarafından desteklenen Ulusal Sağlık Enstitüleri.

1. kaplama yemekleri (1 gün)
Not: parmak izi ya da kir Kalenin dibinde bu işlerken bırakmamaya dikkat edin.
<ol><li>Fare kollajen IV buz çözme ve 3 mL 10 µg/mL kollajen IV çözeltisi hazırlamak için 50 mM HCl (pH 1.3) ile sulandırmak. Not: Kollajen 37 ° C'de çökelti Bu nedenle kollajen çözüm ise çözülme ve bu modülle çalışmanın bir düşük sıcaklıkta tutmak önemlidir. Uygun büyüklükteki aliquots hazırlayarak tekrarlanan donma-çözülme döngüsü önlemek ve onları-80 ° C'de depolama</li>
	<li>12-şey cam alt plakanın her şey için 250 µL kollajen IV çözüm ekleyin ve uygun ölçekli, kapanış sıkı bir plastik kutuya koyun. Islak kağıt havlu üzerinde ve çevresinde plakaları nemli bir odası imalatı ve kutusunu kapatmak yerleştirin. Gecede, oda sıcaklığında plakaları kuluçkaya. Not: olarak sadece büyüme alanı kaplı olması gerekiyor, bu yalnızca alt plaka kollajen çözüm ile bağlı olduğu kapak notu karşılamak için yeterli olur ( şekil 1A, B bir şematik plaka için bakınız).</li>
	<li>Ertesi sabah, iki kez absorbe olmayan kollajen ve tampon kaldırmak için deiyonize H2O ile plakalar durulayın. Su iyi değil (su sıçrama bu alana pipetted ise) plaka alt ve coverslip tarafından kurulmuş kenarına plaka kenarına doğrudan. Tabakalar laminar akış başlıklı kuruması. Tabak hemen kullanın veya 4 ° C'de 5 güne kadar saklayın. Not: Farklı kaplama, fibronektin veya kollajen gibi ben farklı hücre hatları gerektirebilir.</li>
</ol>2. nokta tahlil (2 gün) kaplama
<ol><li>
Hücre süspansiyon hazırlama
	<ol>
<li>Hücre süspansiyon hazırlamak için 60 mm doku kültürü çanak ile % 5 at serum % 80 izdiham için takıma DMEM/F12 orta artış var hücreleri kullanın.</li>
		<li>Kalsiyum ve magnezyum ücretsiz Dulbecco'nın fosfat tamponlu tuz çözeltisi (DPBS) içeren hücreleri bir kez durulama, 500 µL tripsin-EDTA (oda sıcaklığında) ekleyin ve 3-5 dk 37 ° C, % 5 CO2, oksijen atmosfere bir kuluçka için hücreleri kuluçkaya.</li>
		<li>4.5 mL kültür tripsin etkinliğini durdurur ve bir 20 Say için orta müstakil hücrelerde askıya µL aliquot bir hemasitometre hücre süspansiyon.</li>
		<li>Tarafından Santrifüjü 200 x g 3 dk de 15 mL konik tüp içinde kalan hücreleri cips, süpernatant Aspire edin ve kültür orta 3 x 106 hücre/mL olarak hücrelerde resuspend. Not: diğer kaplama Substratları ve/veya hücre satırları için optimum hücre yoğunluğu seyreltme serideki oluşturulmalıdır. 3 x 106 hücre/mL başlangıç noktası olarak önerilir.</li>
	</ol>
</li>
	<li>
Kaplama hücreleri
	<ol>
<li>Yer hücre süspansiyon kollajen IV her kapak kayma ortasındaki bir 10 µL damla 12-şey cam alt plaka dokunmadan veya kaplama (şekil 1A, B) tırmalamak kaplı. 30 dk 37 ° c, oksijen atmosfere hücreleri eklemek izin vermek için plaka kuluçkaya. Bir ters mikroskobu hücreleri bağlı olduğu kontrol edin. Not: Bu en iyi damla, kenarında nerede bir monolayer oluşumu (şekil 1C) gözlenen görülebilir. Gerekirse, plaka kadar kuluçkaya 3 h. damla hızlı bir şekilde kuru olabilir gibi nem kuluçka kadar yüksek olduğunu emin olun. Damla birimin bir azalma bu adımı sırasında gözlenen, PBS nem oranı artırmak için kuyu arasında boşluk ekleyin.</li>
		<li>Yavaşça wells kültür iki kez ekli olmayan hücreleri kaldırmak için orta 1 mL ile yıkayın. 1 mL kültür orta her şey için ekleyin. Bir ters mikroskobu kontrol kayan hiçbir hücre kalır, aksi takdirde kuyuları tekrar yıka. Gecede, 37 ° C, % 5 CO2, oksijen atmosfer iyi tanımlanmış hücre yaprak elde etmek için hücreleri kuluçkaya. Not: Yüzen hücreleri yeniden plakasına eklemek ve istenmeyen hücre kolonileri kurmaya muhtemeldir.</li>
	</ol>
</li>
</ol>3. uyarıcı hücreleri (3. gün)
<ol><li>Bütün wells kolonileri düzgün büyüdü o hücreye sağlamak için ters bir mikroskop kullanarak kontrol edin. Gerekirse, uygun olmayan wells işaretlemek ve bunları kullanmayın.</li>
	<li>Her şey uygun bir şekilde araştırıldı her koşul için plaka etiketleyin. Her koşul yinelenen veya nüsha çalıştırın.</li>
	<li>DMEM/F12 %0,1 at serum (Aç orta) içeren hücrelerde açlıktan için orta değiştirmek 3 h önce hücreleri uyarılır.</li>
	<li>Hücreleri EGF (5 ng/mL nihai toplama) eklenmesi veya istenilen başka arabulucu tarafından teşvik ve karışımı yavaşça 1 mL micropipettor kullanarak orta veya plaka dönen tarafından.</li>
	<li>Plaka kenar deplasman ve kenar kontur 4.1 bölümünde açıklandığı gibi gözlemlemek 1-4 gün kuluçkaya veya 4.2 bölümünde açıklandığı gibi hızlandırılmış görüntüleme gerçekleştirmek.</li>
</ol>4. hücre kolonileri ve hücre göç görselleştirme
<ol><li>
Hematoksilen-koloni büyüme (4-7 gün) ölçmek için boyama Eozin (o)
	<ol>
<li>% 70 etanol (1 mL/de) ~ 2 min için hücrelerde düzeltmek.</li>
		<li>Çekirdeklerin Hematoksilen (1 mL/de), ~ 2 dk ile leke.</li>
		<li>Musluk suyu (1 mL/de) çekirdeği kadar yıkama hücrelerle mavi, 5-10 dak.</li>
		<li>Sitoplazma Eozin (1 mL/de), ~ 2 dk ile leke.</li>
		<li>Deiyonize su (1 mL/de) ile durulayın. Not: Hematoksilen Eozin çözümler toplanan ve boyama zayıf hale gelinceye kadar birden çok kez kullanılan.</li>
		<li>Plaka kuruması.</li>
		<li>Plaka çevirmek ve bir cetvel ile nokta çapı ölçmek. Alternatif olarak, Resimler plaka kamera ile fotoğraf çekip ImageJ nokta çapı analiz.</li>
	</ol>
</li>
	<li>
Hızlandırılmış mikroskobu (3. gün)
	<ol>
<li>Kuluçka makinesi mikroskobunun geçin ve 37 ° c sıcaklık ayarlama Nemlendirici dH2O. küçültme/büyütme CO%2 5 ile doldurun. Kuluçka odası nemlendirilmelidir ve motorlu sahne özgürce hareket edebilirsiniz olun. Kuluçka odası kapatın ve 37 ° C'ye ayarlamak mikroskop izin Not: Mikroskop (malzemeleri tablo görmek) kullanılan hücreleri odak sürüklenen önlemek için yansıma önce burada 37 ° c en az 3 h için ısıtmalı olabilir.</li>
		<li>Plaka kuluçka mikroskop sahnesinde yerini ve sahne liste oluşturma. Görüntü iki karşıt kenarları ve her hücre nokta (şekil 1A) merkezi.</li>
		<li>Görüntüler her 3 dk, 15-24 h 10 X amacı istimal için tutar. Veri indirme ve görüntü analizi tercih, bir programda devam örneğin, ImageJ veya Matlab.</li>
	</ol>
</li>
</ol>

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Yazar ifşa etmek hiçbir şey vardır.

Uzak doku çevreleyen doku istila ve uzak siteleri15' e metastaz kökenli tümör ilerleme hücreleri sırasında geçirilir. Geçiş hücre hücre koloni uzak nokta assay olarak görülebilir. Burada, nokta tahlil insan meme kanseri hücrelerinin yanıt EGF olarak göçmen fenotip analiz ederek gösterilmektedir.
Nokta up birkaç adımda doğru ve yinelenebilir sonuçları elde etmek için deneyleri kritik öneme sahiptir. İlk olarak, kaplama plaka olmadığını ve g...

Geçiş deneyleri yaygın olarak tümör hücreleri vitroinvaziv ve metastatik potansiyeli değerlendirmek için kullanılır. En sık, yara veya karalama tahlil epitel sayfaları geçiş bir hücre seçili alan1,2,3içine değerlendirmek için kullanılır. Kazı-kazan tahlil gerçekleştirmek için hücre monolayer kaplama ve bir "scratch" veya boş hücre alan bir pipet ucu ile oluşturulur. Kazı-kazan tahlil yaygın olarak bulunan doku kültürü malzemeleri ile kurmak kolaydır ve çok iyi Kaplamalar, birden fazla örnekleri işleme için izin, gerçekleştirilebilir. Ancak, sıfırdan yapılmış gibi hücreleri monolayer fiziksel olarak kaldırılır ve sık sık hücre ölümü tabi. Ayrıca, hücre dışı matriks plakasına bağlı kez tırmalamak işlemi sırasında hasar görmüş. Benzer şekilde, silikon kullanımı ekler (Ibidi odaları4gibi) veya şablonlar5,6,7 hücrelerin mekanik bozulma ve kaplama için kullanılan matris proteinlerin kısmi kaldırma yol açabilir tabaklar. Yara ya da çizik kapatılması izleme deneyleri bir diğer dezavantajı kendi sınırlı bir süre ders gibidir çizik kapatılana kadar hücre göç sadece analiz edilebilir.
Nokta tahlil gerçekleştirmek için hücreleri üzerine kaplamalı veya kaplamasız levha8dairesel bir koloni olarak kaplama. Bu kaplama strateji için gerekçe göç veya boş hücre çevresinde kültür hücreleri veya ekler kaldırılmasını tarafından rahatsız etmeden istila tanımlı kenarları hücre yaprak elde etmektir. Nokta tahlil genel amacı geçiş kenar yer değiştirme veya daha yüksek spatio-zamansal çözünürlük hücrelerinin göçmen fenotip çözümlemek için hızlandırılmış görüntüleme için farklı koloni çapı, de tarafından ölçülen hücre yaprak gözlemlemektir.
Hücre göç microenvironmental yardımlar kemokinler, sitokinler ve büyüme faktörleri gibi EGF gibi çeşitli tarafından etkilenebilir. EGF üzerinden bağlayıcı için onun reseptör, EGF reseptör9, biyolojik etkileri giderek artan ve invaziv ve metastatik tümör hücreleri4,9,10davranışını artan bir büyüme faktörüdür. Burada, nokta tahlil incelemek için kullanılır EGF uyarılmış bir insan invaziv, akciğer kolonisi meme kanseri hücre satırı (MCF10CA1a)8,11,12oluşturan hücre göç.

<table><tbody><tr><td>MCF10CA1a cells</td><td>Karmanos Research Institute</td><td>N/A</td><td>cells used for assay</td></tr><tr><td>mouse collagen IV</td><td>BD Biosciences</td><td>354233</td><td>used to coat plates</td></tr><tr><td>trypsin / EDTA</td><td>Thermo Fisher</td><td>25300054</td><td>used to remove cells from tissue culture plates</td></tr><tr><td>DPBS&amp;nbsp;</td><td>Mediatech</td><td>21-031-CV</td><td>used to wash cells</td></tr><tr><td>DMEM / F12</td><td>Thermo Fisher</td><td>11320082</td><td>used as culture medium</td></tr><tr><td>horse serum</td><td>Thermo Fisher</td><td>26050088</td><td>used to supplement culture medium</td></tr><tr><td>12 well glass bottom plate</td><td>MatTek</td><td>P12G-1.5-14-F</td><td>used to grow cells for imaging</td></tr><tr><td>EGF (epidermal growth factor, human recombinant)</td><td>Peprotech</td><td>AF-100-15</td><td>used to stimulate cells</td></tr><tr><td>fatty acid free BSA</td><td>Sigma</td><td>A8806-1G</td><td>used to make buffer for EGF</td></tr><tr><td>hematoxylin</td><td>Sigma</td><td>HHS32</td><td>used to stain colonies</td></tr><tr><td>eosin</td><td>Electron Microscopy Sciences</td><td>26051-10</td><td>used to stain colonies</td></tr><tr><td>37 &amp;#176;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Sanyo</td><td>model MCO-18AIC (UV)</td><td></td></tr><tr><td>Zeiss AxioObserver with incubator chamber and motorized stage</td><td>Zeiss</td><td>model Axio Observer.Z1</td><td>used for time lapse imaging</td></tr><tr><td>ORCA Flash LT sCMOS camera</td><td>BioVision</td><td>C11440-42U-KIT</td><td>used for timelapse imaging in combination with Zeiss AxioObserver</td></tr><tr><td>Metamorph</td><td>BioVision</td><td>MMACQMIC</td><td>software used for time lapse imaging</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>model IX70</td><td>used to count cells and to check colonies in tissue culture</td></tr><tr><td>plastic box</td><td>Lock&amp;amp;Lock</td><td>N/A</td><td>used as humid chamber (any tight closing plastic box that fits the plates can be used)</td></tr></tbody></table>

using the dot assay to analyze migration of cell sheets

Karmaşık organizmalara statik görünse de, onların dokular altında sürekli cirosu vardır. Yaş hücreleri ölür ve vardır yeni hücreleri tarafından yerine hücreleri doku içinde sıkıca orkestra bir şekilde hareket. Tümör gelişimi sırasında bu denge bozulmuştur ve tümör hücreleri epitel yerel microenvironment, uzak siteler için seyahat etmek ve sonuçta uzak sitelerdeki metastatik tümörler oluşturmak için istila kökenli bırakın. Nokta tahlil bir boş hücre alana hücre yaprak hareket net değerlendirmek ve parametreleri hızlandırılmış Imaging'i kullanma hücre göç çözümlemek için bir basit, iki boyutlu Kısıtlamasız geçiş tahlil olduğunu. Burada, nokta tahlil epidermal büyüme Malign meme kanseri hücrelerinin potansiyelini artırmak için bilinen faktörü (EGF), hücre göç yanıt çözümlenecek bir insan invaziv, meme kanseri hücre kültürünü, MCF10CA1a, şekillendirme akciğer koloni kullanarak gösterilmiştir ve hücre göç fenotip değiştirmek için.

Burada sunulan (şekil 1) nokta tahlil invaziv, kullanarak gerçekleştirilen bir modeli sistem olarak meme kanseri hücreleri (MCF10CA1a) oluşturan akciğer koloni. Yeni başlayanlar, yapım o a uygun elinde bile son derece tekrarlanabilir hücre nokta nokta tahlil verimleri ve tahlil (şekil 1D) yürütmek kolay. O boyama veya hızlandırılmış görüntüleme nokta tahlil Birleşik ve sonraki partikül imaj ...

Watch this Scientific Journal Video about Using the Dot Assay to Analyze Migration of Cell Sheets at JoVE.com

Using the Dot Assay to Analyze Migration of Cell Sheets

Geçiş hücre yaprak analiz etmek için nokta tahlil kullanarak

Hücre göç geliştirme, doku bakım ve onarım ve tumorigenesis için çok önemlidir ve büyüme faktörleri, kemokinler ve sitokinler tarafından düzenlenmiştir. Bu iletişim kuralı nokta yöntemi, ekli, uyumlu cep yaprak yanıt microenvironmental ipuçlarını göçmen fenotip değerlendirmek için bir iki boyutlu, serbest geçiş tahlil açıklar.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

using-the-dot-assay-to-analyze-migration-of-cell-sheets

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

6.8K Görüntüleme.  National Institutes of Health. Hücre göç geliştirme, doku bakım ve onarım ve tumorigenesis için çok önemlidir ve büyüme faktörleri, kemokinler ve sitokinler tarafından düzenlenmiştir. Bu iletişim kuralı nokta yöntemi, ekli, uyumlu cep yaprak yanıt microenvironmental ipuçlarını göçmen fenotip değerlendirmek için bir iki boyutlu, serbest geçiş tahlil açıklar.

Video: Using the Dot Assay to Analyze Migration of Cell Sheets

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Toksik Kimyasalların Maruz sonra Endotel Hücre Göç Değerlendirilmesi

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Gelişim Biyolojisi

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

Cep işgali ve geçiş işlemi değerlendirilmesi: Video mikroskop esaslı çizik karşılaştırılması tahlil ve Boyden odası tahlil yara

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Kanser Araştırmaları

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Otomatik bir canlı hücre Imager kullanarak basit geçiş/işgali iş akışı

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Microfabricated Kanallar Hücre Göç Çalışması

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Biyomühendislik

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

Protein fonksiyonu için bir 2DE-Jel Spot: Kronik Lenfositik Lösemide HS1 öğrendim Ders

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Tıp

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Göç, Kemo-Attraction, ve Co-Kültür Tahliller İnsan Kök Hücre Kaynaklı Endotel Hücreleri ve GABAerjik Nöronlar için

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Nörobilim

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

Granül Hidrojel Biyomalzemeler İçinde ve İçine Üç Boyutlu Hücre Migrasyonunun Ölçülmesi

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

Ölçme Hücre Göç Bir Özelleştirilebilir Odası

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

Biyoloji

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Hücresel Hareketin Mekanik Kontrolünün Ve İlgili Sinyal Olaylarının Değerlendirilmesi için Tek Hücreli Durotaksis Tsay

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

The Transwell Migration Assay

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer. To quantify cell migration, a simple assay was developed in 1961 by Dr. Stephen Boyden, which is now known as the transwell migration assay or Boyden chamber assay. This set-up consists an insert which separates the wells of a multiwell plate into top and bottom compartments. Cells whose migration is to be studied are seeded into the top compartment and the chemoattractant solution is placed in the bottom compartment. After incubation, counting the cells in the bottom compartment allows quantification of migration induced by chemoattractants.
This video will review the commonly used experimental set-up for cell migration studies. Then we'll highlight a few key considerations, and outline a generalized protocol for running an experiment involving adherent cells. Lastly, we'll review various adaptations of this set-up currently being used to study different factors that affect migration.

The Transwell Migration Assay and Chemo-attractants

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer.  To quantify cell migration, a simple ...