Name: isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement
Uploaded: 2017-07-26T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

Bu çalışma İsviçre Ulusal Bilim Vakfı (hibe numarası 310030-166313), &quot;Vakıf Marcel Levaillant&quot; ve &quot;Fondation pour la recherche osteo-articulaire&quot; başlıklı &quot;Fondation Suisse reourcheurs sur les maladies musculaires&quot; tarafından desteklendi.

Ortopedik cerrahi sırasında sağlıklı hastalarda cerrahi atık olarak insan kas örnekleri (semitendinöz kaslardan alınan dokular) toplandı. İnsan çalışmasıyla ilgili tüm yöntemler, İsviçre Düzenleyici Sağlık Otoriteleri&#39;nin yönergelerine ve düzenlemelerine uygun olarak ve İsviçre&#39;deki Cenevre Kanton Yetkilileri&#39;nden (Protokol CER n ° 12-259) Komisyon Yasası tarafından onaylanmıştır. . Bilgilendirilmiş ve yazılı onaylar, çalışmaya katılan tüm yetişkinlerden elde edildi. ...

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Yetişkin iskelet kasından insan miyoblastlarının izolasyonu ve kültürü, kas farklılaşması ve kas yenilenmesini incelemek için in vitro bir model sunar. Bu yazıda, insan miyoblastlarının yüksek verimliliğini basit ve maliyet-sınırlı şekilde arındırmaya olanak tanıyan bir protokol sunuyoruz. Buna ek olarak, bu teknik, izole edilen miyoblastların yüzdesi ve miyojenik farklılaşma verimi açısından güvenilir ve tekrarlanabilir sonuçlar sağlar. Nitekim ...

İnsan iskelet kasları, miyojenik öncü hücrelerin kaynaşmasından kaynaklanan kontraktil, çok çekirdekli kas liflerinden oluşan gruplardan oluşur. İskelet kası, miyofibarların plazma membranı (sarcolemma) ile bazal lamina arasında bulunan iskelet kası kök hücreleri olan SC&#39;lerin varlığı nedeniyle hasardan sonra yeniden oluşma kapasitesine sahiptir. Yaralanmamış kasında, SC&#39;ler çoğunlukla sessiz bir durumda bulunur. Mekanik stres veya yaralanmaya tepki olarak, SC&#39;ler aktifleştirilir (miyoblastlar), çoğalır ve yeni miyofiberleri oluşturmak için ya da SC havuzu 1 , 2&#39;yi doldurmak için kendi kendini yenilemek üzere farklılaşır. Yıllar geçtikçe, SC&#39;leri ve yavrularını (miyoblastları) iskelet kası biyopsilerinden izole etmek için çeşitli teknikler geliştirildi. Bu hücreler üzerinde ifade edilen hücre yüzey markörlerinin daha iyi anlaşılması ve floresans ile aktive edilmiş hücre sınıflandırması (FACS) metodolojisine 3 ile, 4 , 5 , şimdi insan kasları saf kas popülasyonlarını kas örneklerinden izole etmek mümkündür. Kalsiyum sinyali iskelet kası gelişimini, homeostazı ve yenilenmeyi düzenler. Özellikle, SOCE adlandırılan hücre-içi stokunun tükenmesi sonra aktive edilir Ca2 + girişi, bu işlemler için büyük önem 6 taşımaktadır. Hücre uyarımı üzerine, endo / sarkoplazmik retikulumdaki Ca 2+ seviyesi (ER / SR) azalır ve bu da, Ca 2+ girişinin ER / SR 7&#39;yi tekrar doldurmasına izin veren plazma membranı Ca 2+ kanallarını harekete geçirir. SOCE&#39;ye katılan iki ana protein, ER / SR Ca 2+ -gören stromal etkileşim molekülü 1 (STIM1) proteini ve plazma membranı Ca2 + kanalı Orai1&#39;dir. İskelet kası bu iki proteini ve aynı ailelerin diğer proteinlerini bol miktarda ifade eder (STIM2 aNd Orai2-Orai3) 8 , 9 ve birkaç Ca 2+ geçirgen olmayan geçici alıcı potansiyel kanonik (TRPC) ailesi kanalları 10 , 11 , 12 , 13 . SOCE yolu boyunca Ca 2+ girişi, kas oluşumu / rejenerasyonu için çok önemlidir 14 . STIM1 veya Orai1 proteinlerinin mutasyonları kas hipotoni 15 esas açan kas fonksiyonu üzerinde zararlı etkileri vardır. SOCE bozukluğu olan hayvan modelleri, azaltılmış kas kütlesini ve kuvvetini, aynı zamanda artmış yorgunluk 6 , 16 , 17 ile birlikte gösterir . Belirtildiği gibi, diğer STIM ve Orai proteinleri ve birçok TRPC kanalı iskelet kasında eksprese edilir ve şimdiye kadar ilgili rolleri belirlenememiştir. D&#39;yi çalarakKendi ifade seviyelerine sahip oldukları için, SOCE&#39;deki etkilerini ve insan iskelet kası farklılaşması sırasındaki rollerini araştırmak mümkündür. SOCE ölçümü iki farklı yaklaşımla yapılabilir: elektrofizyolojik güncel kayıtlar ve Ca 2+ ölçümleri. Elektrofizyoloji, ilgi akımını ve buna bağlı elektrofizyolojik imzayı ölçtüğünden kesinlikle daha doğrudan bir yöntemdir. Bununla birlikte, bu tekniğin kas hücrelerine uygulanması çok zordur çünkü esas olarak kas hücrelerinin büyüklüğü ve endojen SOCE akımının küçük boyutu. Sitosolik Ca 2+ görüntüleme teknik açıdan çok erişilebilir, ancak sitosolde ölçülen Ca 2+ seviyesi hem Ca 2+ girişini hem de hücrenin yeniden dışarı pompalamasını ya da iç depolarda yansıtıldığından önlem daha dolaylı. Mikoblastların küçük insan iskeletinden izole edilmesini içeren bir metodolojiEnzimatik sindirim, amplifikasyon ve FACS sonrası etal kasa bu makalede açıklanmıştır. Zamanla farklılaşan belirteçlerin ekspresyonunu izleyen kas farklılaşması ve immünofloresan protokolü işlemi 18&#39;de tanımlanmıştır. Son olarak, SOCE&#39;nin ölçümü ve Ca 2+ sinyal verme ve iskelet kasının diferansiyasyonunda farklı iyon kanallarının rolü açıklanmaktadır.

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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Ham&#8217;s F10</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">31550-023</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">FCS</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">10270-106</td>
			<td style="width:201px;">Lot 41G5532K</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">BSA</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">O5482</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">fetuin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">F3385</td>
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			<td height="25" style="height:25px;width:321px;">EGF</td>
			<td style="width:235px;">Corning</td>
			<td style="width:187px;">354001</td>
			<td style="width:201px;"></td>
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			<td height="25" style="height:25px;width:321px;">Dexamethansone</td>
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			<td style="width:187px;">D1756</td>
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			<td height="25" style="height:25px;width:321px;">Insulin</td>
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			<td style="width:187px;">I9278</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Creatine</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">27900</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Pyruvate</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">P4562</td>
			<td style="width:201px;"></td>
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			<td height="25" style="height:25px;width:321px;">Uridine</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">U3003</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Gentamycin</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">15710-049</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Trypsin-EDTA 0.05%</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">25300-062</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">70 &#956;m cell strainer</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352350</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">40 &#956;m cell strainer</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352340</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 50&#160;ml tube</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352070</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 15&#160;ml tube</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352096</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 5&#160;ml tube (FACS)</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352058</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Culture medium: OPTI-MEM</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">31985-047</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Transfectant reagnt: RNAiMax</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">1756064</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PBS</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">14190-094</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mounting medium</td>
			<td style="width:235px;">Fluka</td>
			<td style="width:187px;">10981</td>
			<td style="width:201px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti-MyHC *</td>
			<td style="width:235px;">DSHB</td>
			<td style="width:187px;">MF20&#160;</td>
			<td style="width:201px;">concentrate</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti-myogenin</td>
			<td style="width:235px;">BD Pharmigen</td>
			<td style="width:187px;">556358</td>
			<td style="width:201px;"></td>
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			<td height="25" style="height:25px;width:321px;">Rabbit anti-MEF2</td>
			<td style="width:235px;">Santa Cruz Biotech</td>
			<td style="width:187px;">C-21</td>
			<td style="width:201px;"></td>
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			<td height="25" style="height:25px;width:321px;">Goat anti-mouse Alexa488</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">A11029</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Goat anti-rabbit Alexa 546</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">A11035</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD56-Alexa488</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557699</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD146-PECy7</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562135</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD45-PE-CF594</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562279</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD34-APC</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">345804</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD144-PE</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">560410</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Alexa Fluor 488 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557702</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PECy7 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557872</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PE-CF594 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562292</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">APC mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">550854</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PE mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">556650</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">DAPI</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">D9542</td>
			<td style="width:201px;">CAUTION:&#160; toxic compound</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Paraformaldehyde</td>
			<td style="width:235px;">Fluka</td>
			<td style="width:187px;">762400</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Fura-2 AM</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">F1201</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Pluronic F-127</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">P3000MP</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">Thapsigargin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">T9033</td>
			<td style="width:201px;">CAUTION&#160;: toxic compound&#160;</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">Ratio imaging acquisition software: Metafluor 6.3</td>
			<td style="width:235px;">Molecular Device</td>
			<td style="width:187px;"></td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="24">
			<td colspan="2" height="24" style="height:24px;">*&#160;: MF 20 was deposited to the DSHB by Fischman, D.A.&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement

Uydu hücreleri (SC), kas liflerinin plazma membranı ile çevresindeki bazal laminat arasında bulunan kas kök hücreleridir. Kas rejenerasyonu için gereklidirler. İskelet kaslarında sıklıkla görülen yaralanmalar üzerine SC&#39;ler harekete geçirilir. Myoblastlar olarak çoğalıyor ve kas lezyonlarını onarmak için ayırt ediliyorlar. Kas farklılaşması sırasında gerçekleşen birçok olay arasında, sitosolik Ca 2+ sinyalleri büyük önem taşır. Bu Ca2 + sinyaller hem de hücre dışı alanı Ca2 + girişi, özellikle depo çalışan Ca2 + girişi (SOCE) itibaren, iç Ca2 + depolardan Ca + 2 serbest ortaya çıkar. Bu makale, ortopedik ameliyattan sonra toplanan kas örneklerinden saf bir insan miyoblast popülasyonunu elde etmek için kullanılan bir metodu açıklamaktadır. Doku mekanik ve enzimatik olarak sindirilir ve hücreler çoğaltılır ve daha sonra spesifikasyonların varlığına göre akış sitometrisi ile sıralanırFik membran belirteçleri. Elde edilen insan miyoblastları büyütülür ve büyüme faktörlerini kültür ortamından uzaklaştırarak ayırt etmeyi taahhüt eder. Spesifik transkripsiyon faktörlerinin ekspresyon seviyeleri ve in vitro immünofloresans, kontrol koşullarında ve Ca 2 + sinyallemeyle ilgili proteinleri susturmadan sonra miyojenik farklılaşma sürecini değerlendirmek için kullanılır. Son olarak, SACE&#39;nin güvenilir ve tekrarlanabilir ölçümlerini sağlayan bir ratiometric Ca 2+ probu olarak Fura-2&#39;nin kullanımını ayrıntılarıyla açıkladık.

Bir insan kas örneğinin enzimatik ayrışmasından sonra, hücreler GM&#39;de amplifiye edildi. CD56 + / CD146 + / CD45- / CD34- / CD144- hücreleri olarak tanımlanan insan miyoblastları FACS&#39;den sonra elde edildi. Myoblastlar, analiz edilen popülasyonun% 60&#39;ından fazlasını temsil etti ( Şekil 1 ). Primer insan miyoblastları yeniden oluşturuldu, konfluansa kadar çoğaltıldı ve 48 saat DM&#39;de kültürlendi. Transkripsiyon faktörü MEF2 ve kas spesifik protein miyozi...

Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Burada, insan miyoblastlarının saf bir popülasyonunu yetişkin kas dokusundan elde etmek için bir metodoloji tarif ederiz. Bu hücreler, in vitro iskelet kası diferansiasyonunu incelemek ve özellikle Ca 2+ sinyallemeyle ilgili proteinleri incelemek için kullanılırlar.

İnsan Myoblastlarının İzolasyonu, Miyojenik Farklılaşmanın Değerlendirilmesi ve Mağazada Kullanılan Kalsiyum Giriş Ölçümü

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

The overall goal of this procedure is to obtain a pure population of human myoblasts from adult skeletal muscle. These cells are used for functional studies such as assessment of myogenic differentiation and measurement of store-operated calcium entry. The main advantage of this technique is that it allows a rapid, simple and reproducible isolation of human primary myoblasts.These myoblasts are the new ex-vivo to study the calcium signaling during muscle regeneration. This method can help answer key questions in the muscle field such as, identifying the ionic channels and the transcription factors involved during muscle differentiation. For this method can provide insight into gene functions during human myogenic differentiation.It can also be applied to other studies such as, potential for using myogenic stem cells to treat muscular dystrophies. Transfer the muscle sample into a sterile six centimeter plate, wash the sample with PBS twice. Remove any remaining adipose tissue and weigh the sample.Next, add trypsin and mince the muscle into pieces smaller than two millimeters in size. Once minced, use a 10 milliliter pipette to transfer the tissue into a 200 milliliter dissociation bottle containing a stir bar, then continue collecting minced tissue in trypsin solution until the dissociation bottle is filled to 90 milliliters. Now, incubate the tissue in a water bath at 37 degrees celsius for 60 minutes with gentle stirring.After an hour, add 10 milliliters of FCS to stop the reaction and homogenize the mixture using trituration. Now, load 70 micron strainers onto two 50 milliliter tubes and pass half of the cell suspension through each strainer by pipetting the suspension into the filters. Next, make a suspension of the cells in growth medium and plate 200, 000 cells onto six centimeter plates with four milliliters of medium.Incubate the plates, changing the medium every third day. After five to seven days, the cells should be 70%to 80%confluent, then, stain them. To begin, rinse the plate of confluent cells with PBS.Then, add one milliliter of trypsin solution and let the enzyme work for three minutes. Stop the reaction by adding two milliliters of medium and collect the cells in a 15 milliliter tube. Then, spin the cells down and re-suspend them in one milliliter of medium.Next, take a cell count while storing the cells on ice. For the facts analysis, load seven tubes with 100, 000 cells each. Load the remaining cells into an eighth tube for sorting.Wash the cells with one milliliter of cold fax buffer, then, centrifuge the tubes and aspirate and discard the supernatant. Add back 200 microliters of fax buffer with the appropriate antibodies and let the cells incubate on ice for 30 minutes. After 30 minutes, wash the cells again and re-suspend them in 500 microliters of fax buffer, then sort the cells using a flow cytometer.After excluding the CD45 positive hematopoietic cells, separate the CD45 negative cells based on CD56 expression. Then, gate the CD56 positive population for CD34, CD144 and CD146. Re-analyse a fraction of the sorted population, to check the purity.Now, pull the human myoblasts and wash them with five milliliters of medium. Then re-suspend them in one milliliter of medium. Transfer the suspension to a six centimeter plate with four milliliters of medium and incubate the plate at 37 degrees celsius with 7%carbon dioxide.For this procedure, have cultured myoblasts and make fresh transvection medium. Incubate the medium at room temperature for 15 to 20 minutes. During the medium incubation, prepare the myoblasts.First, rinse them once with PBS. Then, trypsinise the adherent cells. Stop the reaction by adding two milliliters of medium.Then, collection the cells into a 50 milliliter tube. Spin them down and re-suspend them in one milliliter of medium. For each transvection, prepare 500, 000 cells in 200 microliters of medium.To each 500 microliters of transvection medium, add a 200 microliter aliquot of cells and then mix using only two strokes of the pipette gun. Then, let the reactions proceed for five minutes at room temperature. After five minutes, transfer the reactions to 3.5 centimeter culture dishes, each containing one glass cover slip.Next, add 1.3 milliliters of medium for a final volume of two milliliters and incubate the dishes. After 48 hours of differentiation, the cells can be immunostained or analyzed using calcium imaging. For either option, first carefully wash the cells twice using one milliliter of room temperature PBS to wash.Try to avoid detaching the myotubes. For a calcium imaging, cover the washed cells with two micromolar fura-2 AM plus one micromolar threonic acid in calcium containing medium. Then, let them incubate for about 30 minutes before mixing.Next, wash the cells twice with unmodified calcium containing medium and then, incubate them for 10 to 15 minutes for the esterification. Now, remove the cover slip from a culture plate and install it into the experimental chamber. Load the chamber with calcium containing medium, place it under a microscope, then, start the data recording.Fura-2 is excited alternately at 340 and 380 nanometers. The emission light is collected at 510 nanometers. Diving the 340 nanometer nanometer images by the 380 images, provides the image ratios as the fluctuations and calcium concentration are relatively low, acquire one image ratio every two seconds.After two or three minutes, replace the medium with calcium free medium. Then, for one or two minutes, monitor the activity of the store operated calcium in the cells. Next, add one micromolar thapsigargin to the cells to deplete their internal calcium stores.Wait eight to 10 minutes, then, rapidly replace the medium with the calcium containing medium and record for five minutes while calcium enters the cells via the suc e channels. After terminating the experiment, analyze the images. Select an area where there are no cells for region one, which defines the background, then, select an area in the cytoplasm for region two.The two regions can be drawn on any of the images in the series. Next, go to the run experiments tab and select reference images for the 340 and 380 channels, select region one, then click on subtract background. Now, run the entire experiment to remove the background from all the images.Once completed, click log data option to open a spreadsheet in which the time and the ratio data are logged. Muscle cells were grown up, expanded and sorted using fax. Myoblasts represented more than 60%of the analyzed population.The primary human myoblasts were then cultured in differentiation medium and immunostained for the transcription factor of MEF2 and myosin heavy chain proteins. A majority of cells expressed myosin heavy chain and formed myotubes with fusion index values of about 60%the other 40%of the cells remained undifferentiated mononucleated cells, also known as reserve cells. The described store operated calcium entry experiment were successfully performed on the differentiated cells.Performing the same experiment to on cells transvected with small interfering RNA, directed against calcium permeable channels of the TRPC family, shows that the knockdown had a measurable impact on store operated calcium entry. After watching this video, you should have a good understanding of how to isolate human myoblasts from adult skeletal muscle. We use these cells to study the successive steps of muscle differentiation and in particular, deactivation of the transcription factors and calcium channels.Once mastered, this technique can be done in less than three hours if it is performed properly. It is a rapid, simple, reproducible method that obtains the yield of human myoblasts. While attempting this procedure, it is important to prevent the myoblast cultures from reaching full confidence, as there will be unwanted spontaneous differentiation.

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

İnsan İskelet Kası İlköğretim andMyogenic Hücreleri ve Fibroblast İzolasyon ve Kantitatif İmmünositokimyasal Karakterizasyonu

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Evans Blue Dye ile Zebra balığı Larva İskelet Kası Bütünlüğü Analizi

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

SH-SY5Y İnsan Nöroblastom Hücre Hattı Farklılaşma

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Domuz Subkutan yağ dokularındaki İzolasyonu ve Yağ-Türetilmiş Kök Hücre Farklılaşması

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

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

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 ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

İlkel ve kesin hematopoetik ataları insan Pluripotent kök hücrelerden farklılaşma yönetmen

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Yalıtım, kültür ve kemik iliği Stromal hücreler ve Osteoklast ataları fareler gelen farklılaşma

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Insan pluripotent kök hücrelerinin karaciğer hücrelerine verimli farklılaşma

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

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Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...