T. Fritsch, A. kurt-Asseburg, ı. Linde ve S.-M teşekkür ederiz. Tokarski mükemmel hayvan bakım ve teknik destek için. Yoğun ve yararlı tartışmalar için Köster laboratuvar Tüm üyeler için minnettarız. Deutsche Forschungsgemeinschaft (KO 1949/5-1) ve Federal Devlet Aşağı Saksonya, Niedersächsisches Vorab (VWZN2889) tarafından finansman minnetle anıyoruz.

<ol>
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Yazarlar ifşa gerek yok.

Burada, iki farklı protokol 2 dpf zebra balığı embriyo veya yetişkin zebra balığı beyin Kültür primer hücre için mevcut.
2 dpf zebra balığı birincil hücre kültürleri hazırlanması temel hücre kültürü teknikleri deneyimi olan herkes için gerçekleştirmek nispeten kolaydır. Ancak, iyi ve tekrarlanabilir sonuçlar elde etmek için yeterli sayıda embriyo malzeme başlangıç olarak çok önemlidir (100 olduğunu en azından). Embriyo yükseltme sırasında tüm olası kaynaklarını kirlenme mikroplar ve parazitler miktarını mümkün olduğunca düşük tutmak için kaçınılmalıdır. Kuluçka embriyoların etanol içinde en kritik — bu adım ayrıntılı Sterilizasyon olun ama etanol hücre ve doku zarar organik bir çözücü olduğu için pek de uzun kadar uzun olması gerekiyor.
Zamanlama sırasında beyin doku veya 2 dpf zebra balığı embriyo ayrılma enzimatik esastır. Kuluçka süresi aşılmaması ve tek hücreli aşamaya tam ayrılma başarılı bir kez enzim içeren orta hızlı bir şekilde çıkarmak. Yine de, tip 2 collagenase hücre zarı zarar vermez ve değil güçlü olarak FBS, tripsin33olarak tarafından engellenir. Bu özellikler sayesinde, biz daha yüksek verim ve hassas hücre tiplerinin canlılığı ile sonuçlanan FBS huzurunda ayrılma hücre canlılığı desteklemek ve hücre hasarı, azaltılması önemli bir ek olarak gerçekleştirmek başardık.
Temsilcisi sonuçlarımız tarafından gösterildiği gibi farklı hücre tipleri de morfoloji veya hücre türüne özgü transgenes floresan muhabirleri kodlama ifade tarafından tanımlanan olabilir. İkinci canlı hücre görüntüleme belirli hücre nüfus19gerçekleştirirken çok uygundur. Ancak, tutarlı sonuçlar sağlamak için floresans mikroskobu tarafından 2 dpf embriyo ilgili artırıcı ifade desenini doğrulamak için önerilir.
Embriyonik zebra balığı primer hücre hücre altı yapılarını görüntüleme için izin vermek için bir adım Protokolü floresan organel işaretleri bir seçim için kodlama plazmid DNA transfect kurduk. Bu yöntemle transfected hücre oranı nispeten düşük ama güvenilir ve yeterli hücre sayıları19görüntüleme canlı hücre için sağlar. Başarılı bir transfection için süspansiyon hücre sayısı doğru belirlenmesi önemli bir adımdır. Ortalama içinde 106 hücre yaklaşık 90 2 dpf embriyo elde edilebilir, ancak doğru hücre önceki adım sayma zorunlu19. Ancak, Cos-7 veya HeLa gibi ortak memeli hücre hatları ile karşılaştırıldığında, birincil hücrelerden 2 dpf zebra balığı embriyo boyutunda çok çeşitlidir, ama ortalama (özellikle nöronlar) oldukça küçük, hangi Neubauer odasında nispeten zor sayım yapabilirsiniz. Aynı sonraki görüntüleme uygulamaları için geçerlidir. Tatmin edici bu küçük zebra balığı hücrelerdeki hücre altı yapıları görselleştirmek için yüksek çözünürlüklü bir Gelişmiş floresans mikroskobu tercihen mikroskobu tarama confocal lazer gereklidir.
Eğitimi gerektirdiğinden primer hücre--dan yetişkin zebra balığı beyin hazırlanması daha gelişmiş hayvan anatomi ve beyin dokusu14ayıklanıyor. Buna ek olarak, Steril koşullar zaten ayıklanan beyin kirletmek değil steril tezgah dışında muhafaza edilmesi gerekir. Ancak, sonraki adımları kaplama ve ayrılma ile ilgili ilk protokole karşılaştırılabilir ve diğer dokulara kas ya da karaciğer gibi yetişkin balık da uygulanabilir.
Her iki iletişim kuralları için ana hücre tipleri kültür, nöronlar gibi yetiştirmek zor sınırlı canlılığı kısıtlamadır. 3 standart koşullar kullanarak dap sonra embriyonik zebra balığı primer hücre yoğunluğu şiddetle böylece deneyler19görüntüleme için zaman aralığı sınırlama azalır. Çoğunlukla fiboblast benzeri hücreler özel olarak uyarlanmış expedients yokluğunda hayatta.
Karmaşıklığı hücre tiplerinin kültüründe survival oranı artırmak için başarılı bir şekilde iyi'nın L-15 orta test ettik. Daha fazla nöron teşvik katkı B-27 nöronal belirli kültür için ile desteklenmiştir. Nöronal hücre hücre kültüründe ayırt etmek ve birkaç gün için hayatta kalmak için gözlemlenmiştir ne zaman tüm embriyo elde edilen (Şekil 2F-ben) yanı sıra--dan yetişkin beyin gibi (Şekil 4E). Buna ek olarak, primer hücre yüksek yoğunlukları (Örneğin, 0,25 x 10 100 mm2başına6 hücre), kaplama düşük yoğunlukları (G. Russo, ilk sonuçlar) aksine hücre canlılığı artırır.
Özel kültür medya ve takviyeleri kullanımı, biz nasıl farklı yüzeylerde kullanımı son kültürü oluşturmaktadır ve seçime bağlı parametre olarak bir alt kümesini hücre büyümesini teşvik kullanılan hücre tipleri çeşitli üzerinde doğrudan bir etkisi vardır rapor türleri. Güçlü fibroblast benzeri hücre adezyon ve nükleer silahların yayılmasına karşı (Şekil 1E, F) kolaylaştırmak için doku kültürü tedavi plastik poli-L-lizin kaplama için kıyasla görünüyor. Aynı zamanda hücreleri bağlı çok sayıda hücre türleri destekler ve belirli adezyon molekülleri (gibi nöronlar) düzgün bağlı değil, büyümek, ya da üzerinde işlem görmüş plastik ayırt etmek ama özel yapıştırıcı destek tercih ederim. Bu yüzden öneririz, ne zaman bir fibroblast veya epitel-benzeri kültür33, hücre kültürü yemekleri kaplama için poli-L-lizin veya diğer hücre özel yüzeylerde kullanmak için almak üzere tasarlanmamıştır.
Daha fazla olabilir zebra balığı in vivo çalışmalar tamamlayacak bir geliştirilmiş ve belirli hücre tipleri ekimi için uyarlanmış ve birçok çeşitli uygulama için kullanılan gibi bizim Protokolü birincil hücre kültürü kullanımı için bir başlangıç noktası göz önünde bulundurun.

Zebra balığı (Danio rerio, D. rerio) temel ve Biyomedikal araştırma1çok sayıda alanlar için omurgalı popüler bir modeldir. Zebra balığı embriyolar rahimhızla geliştirmek, şeffaf ve böylece canlı bir organizma olarak omurgalı geliştirme eğitim mükemmel önkoşulları sağlayan bir mikroskop altında Sığdır. Zebra balığı2genetik tractability nedeniyle, birçok istikrarlı transgenik muhabir satırları hücre türüne özgü ifade çeşitli floresan işaretleri ile belirli hücre popülasyonlarının gözlem için izin kurulmuştur. Zebra balığı topluluk sentetik Kal4TA4 (veya KalTA3-eşdeğer GalFF) ifade bir transgene taşımaya sözde Gal4-sürücü satırları geniş sunar gen Gal4 DNA'ya bağlanıcı etki alanı Maya ile erimiş viral transkripsiyon harekete geçirmek hücre türüne özgü arttırıcılar kontrolü altındaki etki alanları. Bu sürücü satırları transgenes bir muhabir gen erimiş bir tanımlanmış ters yönde harekete geçirmek sırasında (UAS) oluşan taşımak efektör satırlar için üzerinden geçilen. Kal4TA4 protein böylece hücre türü Seçici ifade muhabir gen3,4aktive UAS öğesine bağlar. Hemen hemen tüm kullanılabilir artırıcı ve muhabir'ndaki öğelerin çift transjenik hayvanlar çok çeşitli Kombinatorik çalışmaları için bu yaklaşım sağlar.
Ancak, tek tek hücreler veya hücre altı içeriklerini odaklanmak ile derinlemesine canlı görüntüleme bir bütün ve sürekli değişen embriyo sınırlıdır. Belirli hücre biyolojik soruları ile en yüksek çözünürlük için hücre kültürlerinde kullanımı genellikle tercih edilir. Zebra balığı, bazı hücre satır yok, ama ağırlıklı olarak seçilen5,6,7 olarak kabul edilir ve onların yayılma kez zaman alır. Ayrıca, tüm kullanılabilir hücre satırlarını türetilmiş, fibroblast hücreleri bir tür hücre kültürü kullanarak deneyler sınırlama vardır. Bu nedenle, biz doğrudan doğruya--dan zebra balığı embriyo ve yaklaşımlar ve uzun ömürlü kültür artırmak ekili çeşitliliği genişletmek için birlikte yetişkin zebra balığı beyin primer hücre hazırlamak için hem bir etkili ve kullanımı kolay iletişim kuralı kurulan hücre tipleri. Buna ek olarak, biz embriyonik primer hücre ifade yapıları floresan organel işaretleri ile transfect için bir yordam mevcut. Böylece, hücresel türleri Morfoloji ve hücre altı yapıları korumak onların temel özellikleri farklı hücre tipleri yüksek zamansal ve mekansal çözünürlükte ile çözümlenebilir.

<table>
	<tbody>
		<tr>
			<td>Fish lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AB (wild-type)</td>
			<td></td>
			<td></td>
			<td>established by Streisinger and colleagues, available from the Zebrafish International Resource Center (ZIRC)</td>
		</tr>
		<tr>
			<td>Tg(ptf1a:eGFP)jh1</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which the enhancer of the zebrafish gene ptf1a drives expression of the fluorescent protein EGFP (Parsons et al., 2007)</td>
		</tr>
		<tr>
			<td>Tg(XITubb:DsRed)zf148</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which the Xenopus neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed&#160; (Peri and N&#252;sslein-Volhard, 2008)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>model 5804 R</td>
		</tr>
		<tr>
			<td>ChemiDoc MP imaging system</td>
			<td>BioRad</td>
			<td></td>
			<td>model XRS+, used to acquire black-and-white images of Petri dishes containing 1 da embryos</td>
		</tr>
		<tr>
			<td>confocal laser scanning microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>model SP8, equipped with 28 &#176;C temperature box and a 63 x objective</td>
		</tr>
		<tr>
			<td>epifluorescent microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>model DM5500B, equipped with 28 &#176;C temperature box and a 40 x objective</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell with capacitance extender</td>
			<td>BioRad</td>
			<td>1652661</td>
			<td>electroporation device</td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>GFL</td>
			<td></td>
			<td>model 3011</td>
		</tr>
		<tr>
			<td>incubator for cell culture (28 &#176;C)</td>
			<td>Memmert</td>
			<td></td>
			<td>model incubator I</td>
		</tr>
		<tr>
			<td>incubator for embryos (28 &#176;C)</td>
			<td>Heraeus</td>
			<td></td>
			<td>type B6120</td>
		</tr>
		<tr>
			<td>light microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>model TELAVAL 31</td>
		</tr>
		<tr>
			<td>micro pipettes</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sterile work bench</td>
			<td>Bio Base</td>
			<td></td>
			<td>with laminar flow and UV light</td>
		</tr>
		<tr>
			<td>tweezers</td>
			<td>Dumont</td>
			<td></td>
			<td>Style 5, Inox</td>
		</tr>
		<tr>
			<td>vertical tube rotator</td>
			<td>Labinco B.V.</td>
			<td></td>
			<td>model LD-79</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image Lab Software</td>
			<td>BioRad</td>
			<td></td>
			<td>for the ChemiDoc MP imaging system from BioRad</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for counting 1 dpf embryos by applying the Count particles-tool to the respective black-and-white images; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/. (1997-2016).</td>
		</tr>
		<tr>
			<td>LAS X</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>for both confocal and epifluorescent microscopes from Leica Microsystems</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCS-DCX-tdTomato</td>
			<td>K&#246;ster Lab</td>
			<td># 1599</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-eGFP</td>
			<td>K&#246;ster Lab</td>
			<td># 7</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-H2B-mseCFP</td>
			<td>K&#246;ster Lab</td>
			<td># 2379</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-mClover</td>
			<td>K&#246;ster Lab</td>
			<td># 3865</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-MitoTag-YFP</td>
			<td>K&#246;ster Lab</td>
			<td># 2199</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-ss-RFP-KDEL</td>
			<td>K&#246;ster Lab</td>
			<td># 4330</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-VAMP1-mCitrine</td>
			<td>K&#246;ster Lab</td>
			<td># 2291</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pSK-UAS:mCherry</td>
			<td>K&#246;ster Lab</td>
			<td># 1062</td>
			<td>based on the pBluescript-backbone of Stratagene</td>
		</tr>
		<tr>
			<td>Plasmid numbers refer to the database entries of the K&#246;ster lab. Plasmids are available upon request.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plastic and glass ware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon Cell Strainer (40 &#181;m)</td>
			<td>FALCON</td>
			<td>REF 352340</td>
			<td>distributed by BD Bioscience, used as &#8220;landing net&#8221; to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium</td>
		</tr>
		<tr>
			<td>1.5 mL reaction tubes</td>
			<td>Sarstedt</td>
			<td>72690550</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Sarstedt</td>
			<td>83.3922</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL falconic tube</td>
			<td>Sarstedt</td>
			<td>62.547.004</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sarstedt</td>
			<td>83.3924.005</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyStrainer (40 &#181;m)</td>
			<td>Greiner Bio-One</td>
			<td>542 040</td>
			<td>with venting slots; used to filter cells after collagenase-mediated dissociation</td>
		</tr>
		<tr>
			<td>electroporation cuvette (0.4 cm)</td>
			<td>Kisker</td>
			<td>4905022</td>
			<td></td>
		</tr>
		<tr>
			<td>glass coverslips</td>
			<td>Heinz Herenz Medizinalbedarf GmbH</td>
			<td>1051201</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher Scientific (Menzel Gl&#228;ser)</td>
			<td>631-0845</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Henneberg-Sander GmbH</td>
			<td>9020-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (plastic; 3 mL)</td>
			<td>A. Hartenstein</td>
			<td>PP05</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (plastic; diameter 10 cm)</td>
			<td>Sarstedt</td>
			<td>821473</td>
			<td>for zebrafish embryos</td>
		</tr>
		<tr>
			<td>pipette tips</td>
			<td>Sarstedt</td>
			<td>Blue (1000 &#181;l): 70762; Yellow (200 &#181;l): 70760002; White (10 &#181;L): 701116</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile cell culture dishes (plastic; diameter 3 cm)</td>
			<td>TPP Techno Plastic Products AG</td>
			<td>93040</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile cell culture dishes (plastic; diameter 6 cm)</td>
			<td>Sarstedt</td>
			<td>72690550</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile Petri dishes (plastic; diameter 10 cm)</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td>for brain dissection</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Roth</td>
			<td>0601.1</td>
			<td></td>
		</tr>
		<tr>
			<td>4 % paraformaldehyde in 1 x PBS</td>
			<td>Sigma-Aldrich</td>
			<td>16005</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium nitrate tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1396</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol p.a. 100%</td>
			<td>Sigma-Aldrich</td>
			<td>46139</td>
			<td></td>
		</tr>
		<tr>
			<td>goat &#945;-mouse IgG (Fc specific) FITC conjugated</td>
			<td>Thermo Fisher Scientific</td>
			<td>31547</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Roth</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>high vacuum grease</td>
			<td>DOW CORNING</td>
			<td>3826-50</td>
			<td>silicon grease used for self-made glass bottom dishes</td>
		</tr>
		<tr>
			<td>magnesium sulfate heptahydrate</td>
			<td>Merck</td>
			<td>105886</td>
			<td></td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>Serva</td>
			<td>29198.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Tubulin, Acetylated antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua-Poly/Mount (mounting medium)</td>
			<td>Polyscience</td>
			<td>18606</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Biochrom</td>
			<td>L 7240</td>
			<td></td>
		</tr>
		<tr>
			<td>potasssion chloride</td>
			<td>Merck</td>
			<td>104938</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>Roth</td>
			<td>68514-61-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-X Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>T7471</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Synonym: Ethyl 3-aminobenzoate methanesulfonate</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BioRad</td>
			<td>1610407</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco by Life Technologies</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase (Type 2)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17101015</td>
			<td>dissolve powder in cell culture medium (8 mg/mL) and sterile-filter the solution, store aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>pronase (from Streptomyces griseus)</td>
			<td>Roche</td>
			<td>11459643001</td>
			<td>distributed by Sigma-Aldrich, dissolve in 30% Danieau (10 mg/mL) and store aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Medium and solutions for cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 x PBS (Dulbecco&#39;s Phosphate Buffered Saline)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-169</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>CO2-independent medium</td>
			<td>Gibco by Life Technologies</td>
			<td>18045054</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>filtrated bovine serum (FBS)</td>
			<td>PAN-Biotech</td>
			<td></td>
			<td>individual batch</td>
		</tr>
		<tr>
			<td>glutamine 100 x</td>
			<td>Gibco by Life Technologies</td>
			<td>25030081</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium</td>
			<td>Gibco by Life Technologies</td>
			<td>11415049</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>PenStrep (10,000 units/mL)</td>
			<td>Gibco by Life Technologies</td>
			<td>15140148</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
	</tbody>
</table>

culture and transfection of zebrafish primary cells

Zebra balığı embriyo şeffaf ve hızla böylece bir sağlam ve gelişmekte olan omurgalılar dinamik biyolojik süreçlerin mükemmel vivo içinde görüntüleme için izin Anne, dışarıda geliştirmek. Ancak, farklı hücre tipleri ve hücre altı yapıları türleri morfoloji ayrıntılı görüntüleme tüm bağlar sınırlıdır. Bu nedenle, etkili ve kullanımı kolay bir iletişim kuralı kültür canlı primer hücre için zebra balığı embriyo ve yetişkin doku kurduk.
Kısaca, dechorionated, deyolked, steril ve collagenase sahip tek kişilik hücrelerin ayrışmış 2 dpf zebra balığı embriyo vardır. Filtrasyon adımdan sonra primer hücre cam alt yemekleri kaplama ve birkaç gün için ekili. Taze kültürler, olduğu kadar uzun vadeli differenciated olanlar, yüksek çözünürlüklü confocal görüntüleme çalışmaları için kullanılabilir. Kültür çizgili miyositler ve poli-L-lizin kaplama üzerinde önemli varlık nöronlar ile farklı hücre türleri içerir. Özellikle etiket hücre altı yapıları için floresan marker proteinler tarafından plazmid DNA nöronlar da dahil olmak üzere farklı hücre tipleri, içine transfection sağlayan bir adım iletişim kuralı da kurduk. Böylece, operatör huzurunda tanımlanmış uyaranlara, karmaşık hücre davranış ve hücre içi birincil zebra balığı hücre dinamiği yüksek uzamsal ve zamansal çözünürlük ile değerlendirilebilir. Buna ek olarak, Yetişkin zebra balığı beyin kullanarak, açıklanan ayrılma tekniği gibi temel kodlamayla koşulları da yetişkin zebra balığı doku için iş göstermektedir.

Şekil 1G çizgili miyositler ve en bol olan nöron benzeri hücre kümeleri ile vahşi türü embriyo elde tipik bir kültürün iletilen bir ışık görüntü gösterir. Bazı hücre tipleri daha kolay tanımlamak için hücre türüne özgü ifade floresan bir protein ile transgenik bir çizgi olabilir (Şekil 1 H) kullanılır.
PCS2 + transfection-floresan protein (eGFP)15 wild türü embriyo sonuçlar 1 güçlü bir floresan sinyal primer hücre içine dap gelişmiş yeşil kodlama plazmid11 (Şekil 2A) dayalı. Tarafından transfecting pCS2 +-floresan organel işaretleri endoplazmik retikulum hedefli kırmızı floresan protein (ss-RFP-KDEL)16 veya mitokondri hedefli Sarı floresan protein (MitoTag-YFP)17, gibi kodlama plazmid dayalı 18, hücre altı yapıları (Şekil 2B, C) ayrıntılı olarak görüntülenir. En çok üç plazmid Co transfection sağlar aynı hücre19 MitoTag-YFP gibi Mikrotubul marker ile birlikte çeşitli floresan füzyon proteinlerin hücre altı yerelleştirme eşzamanlı analizi için insan Doublecortin (DCX) 20 kırmızı floresan protein tdTomato21 ' e erimiş ve nükleer marker histon H2B Camgöbeği bir floresan protein (H2B-mseCFP)22,23 (Şekil 2B) erimiş. Hücre altı yapıları da floresan protein24 mCitrine erimiş yüksek zamansal ve mekansal çözünürlüklü, örnek veziküller membran işareti. sivilce ilişkili membran proteini 1 olumlu hareketi (VAMP1) görüntüsü , 25 (Şekil 3). Zaman içinde morfolojik değişiklikleri kolayca kes26 (Şekil 2 g, ben) gibi parlak bir floresan protein ifade tarafından tüm cep telefonu etiketleme tarafından görülebilir. Ancak, her iki Gal4 sürücü taşıyan çift transgenik embriyo floresan protein mCherry27 UAS öğeye primer hücre erimiş kırmızı kodlama pBluescriptSK omurga dayalı bir plazmid başarılı transfection türetilmiş yapý ve bir UAS efektör inşa gösterir Elektroporasyon protokolü de Kombinatorik genetik (2E rakam) için uygundur. Ayrıca, sabit primer hücre kolayca ayirt ve organel özgü floresan boyalar (Şekil 2F, H) kullanarak boyanabilen.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57872/57872fig3.jpg"> Şekil 3: zamanlı hızlandırılmış görüntü hücre altı yapıları. Vahşi türü hücre (dap 1) adet-VAMP1-mCitrine ile transfected. Zaman sukut yazmak çerçevelerin seçili (1 kare her 1,68 alınmıştır s) gösterilir. Ok uçları ve parça renkler göre içinde üç ayrı VAMP1 pozitif veziküller hücre altı dinamikleri vurgulayın. Ölçek çubuğu 10 µm =. Zaman atlamalı mikroskobu tarama confocal lazer kullanılarak kaydedildi. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57872/57872fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
Ayrıca yetişkin zebra balığı dokusu ile ayrılma teknik ve temel kültür koşulları uygulanabilir. Burada, vahşi türü zebra balığı yetişkin beyin dokusundan test ettik. Şekil 4A -D karmaşık nöronal benzeri ağa başlangıçta enkaz kaplı kültürünün ilerici geliştirme gösterir. Yetişkin beyin transgene Tg taşıyan bir Transgenik balık kullanarak (XITubb: DsRed) zf14828tamamen farklı, DsRed ifade nöronlar (Şekil 4E) yakın ayrıntılı olarak incelendiğinde.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57872/57872fig4.jpg"> Şekil 4: yetişkin zebra balığı beynin birincil hücre kültürü. Primer Hücre parlak alan görüntüleri elde edilen yetişkin zebra balığı beyin (a)1 dap, (B) 3 dap, (C) 5 dap ve (D) 8 dap. 1-3 dap, ölü hücreleri ve doku enkaz iken tek veya kümelenmiş nöronal hücre daha belirgin hale kaybolur. 3 dap, nöronal hücre ilk kısa neurites kurmaya başlar. 5'ten itibaren dap, bir ağ ile yüz-in de uzun neurites oluşumu gözlemlemek mümkündür. Ölçek çubukları = 50 µm. hücreleri poli-L-lizin-kaplı bir 96-şey plaka kültürlü. 7 (E) kültürlü hücreleri transgene Tg ifade bir Transgenik balık yetişkin beyinden dap (XITubb: DsRed) zf148. DsRed farklı nöronlar28ifade edilir. Ölçek çubuğu = 100 µm. hücreleri bir poli-L-lizin-kaplı 24-şey tabak içinde kültürlü. Tüm görüntüleri bir epifluorescent mikroskobu tarafından elde. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57872/57872fig4large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

Culture and Transfection of Zebrafish Primary Cells

Biz birincil hücre kültürleri zebra balığı embriyo transfection ve canlı hücre görüntüleme gibi primer hücre--dan yetişkin zebra balığı beyin hazırlamak için bir protokol için hazırlanması için etkili ve kullanımı kolay bir protokol mevcut.

Kültür ve zebra balığı primer hücre Transfection

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

culture-and-transfection-of-zebrafish-primary-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.3K Görüntüleme.  Braunschweig University of Technology. Biz birincil hücre kültürleri zebra balığı embriyo transfection ve canlı hücre görüntüleme gibi primer hücre--dan yetişkin zebra balığı beyin hazırlamak için bir protokol için hazırlanması için etkili ve kullanımı kolay bir protokol mevcut.

Video: Culture and Transfection of Zebrafish Primary Cells

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Yetişkin balığı beyin türevli Neurospheres İzolasyon ve Kültürü

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Gelişim Biyolojisi

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

Japon Bıldırcın Embriyolar Yolk Sac Membran Primary Endodermal Epitel Hücre Kültürü

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

İzolasyon ve Zebra balığı Embriyolar gelen Tek Hücre Karakterizasyonu

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Oldukça Tekrarlanabilir Küçük Molekül Tabanlı Farklılaşma Protokolü kullanarak insan pluripotent kök hücreleri gelen kardiyomiyositlerde Büyük Ölçekli Üretim

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Yetişkin ve Yaşlı İnsanların İskelet Kası miyojenik Öncü Hücreler / İlköğretim miyoblastların hazırlanması ve Kültür

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Yatay Dağ Dağı: Derinin kalın, üç boyutlu doku kesitleri için yeni bir İşleme ve Görüntüleme Protokolü

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Yaşam sağlam Zebra balığı tek hücreli Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

Yalıtım ve kültür insan yetişkin ve Fetal kalp örnekler elde edilen birincil Epicardial hücre

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Çekirdeklerin dağıtım, proteinler ve sitoiskeleti çalışmaya Caenorhabditis elegans Germline sayısal analizi

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

İndüksiyon ve birincil insan hücrelerinde hücresel yaşlanma doğrulama

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...