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

이 작품은 스위스 국립 과학 재단 (부여 번호 310030-166313), &quot;Fondation Suisse la recherche sur les les maladies musculaires&quot;, &quot;Foundation Marcel Levaillant&quot;및 &quot;Fondation pour la recherche ostéo articulaire&quot;에 의해 지원되었습니다.

건강한 환자의 정형 외과 수술 중에 수술 용 폐기물로 인간 근육 샘플 (반결질 근육에서 얻은 조직)을 채취했습니다. 인체 연구와 관련된 모든 방법은 스위스 규정 보건 당국의 지침 및 규정에 따라 수행되었으며 스위스 제네바 당국 (Commission Cantonale d&#39; Ethique de la Recherche)의 승인을 받았다 (프로토콜 CER 번호 12-259) . 이 연구에 참여한 모든 성인 피험자로부터 정보 및 서면 동의를 얻었습니다. ...

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성체 골격근으로부터의 인간 myoblasts의 분리 및 배양은 근육 분화 및 근육 재생을 연구하는 in vitro 모델을 제공합니다. 이 논문에서, 우리는 간단하고 비용 - 제한된 방식으로 인간 myoblasts의 높은 수율의 정화를 허용 프로토콜을 제공합니다. 또한,이 기술은 고립 된 myoblasts의 비율과 myogenic 차별화의 효율성 측면에서 신뢰성과 재현성 결과를 제공합니다. 사실, 우리?...

인간 골격근은 근원 전구 세포의 융합으로 인한 수축성 다핵 근육 섬유 군으로 구성됩니다. 골격 근육은 근섬유 (원형질막)의 원형질 막과 기저 층 (basal lamina) 사이에 위치한 골격근 줄기 세포 인 SC의 존재로 인해 손상 후 재생성 할 수 있습니다. 손상되지 않은 근육에서 SC는 주로 정지 상태에 있습니다. 기계적 스트레스 나 부상에 반응하여 SC는 활성화되고 (myoblast), 증식하고, SC pool 1 , 2 를 보충하기 위해 새로운 myofibers를 형성하기 위해 분화되거나 자체 갱신됩니다. 수년에 걸쳐 골격근 생검에서 SC 및 그 자손 인 myoblast를 분리하기위한 몇 가지 기술이 개발되었습니다. 이 세포들에 표현 된 세포 표면 마커들과 형광 - 활성화 세포 분류 (FACS) 3 의 방법론에 대한 더 깊은 이해로, 4 , 5 , 이제 근육 샘플로부터 인간 근섬유의 순수 집단을 분리하는 것이 가능합니다. 칼슘 시그널링은 골격근 발달, 항상성 및 재생을 조절합니다. 특히, SOCE라는 세포 내 저장 고갈 다음 활성화되는 칼슘 2 + 항목은 이러한 프로세스에 대한 중요성 6이다. 세포 자극시 endo / sarcoplasmic reticulum (ER / SR)의 Ca 2+ 수준이 감소하고 세포막 칼슘 이온 채널을 활성화시켜 Ca 2+ 유입을 허용하여 ER / SR 7 을 충전합니다. SOCE에 관여하는 두 가지 주요 단백질은 ER / SR Ca 2+ - 감지 간질 상호 작용 분자 1 (STIM1) 단백질과 원형질막 Ca 2+ 채널 Orai1입니다. 골격 근육은이 두 단백질과 같은 가족의 다른 단백질을 풍부하게 표현합니다 (STIM2 and Orai2-Orai3) 8 , 9 및 transient receptor potential canonical (TRPC) 계열 10 , 11 , 12 , 13의 Ca 2+ 투과성 채널. SOCE 경로를 통한 Ca 2+ 유입은 근육 형성 / 재생에 매우 중요합니다 14 . STIM1 Orai1 또는 단백질 돌연변이 근육 긴장 저하 (15)을 중심으로 이어지는 근육 기능에 해로운 영향을 미친다. SOCE 손상이있는 동물 모델은 또한 근육 질량과 힘을 감소시키고, 피로도를 6 , 16 , 17 로 향상시킵니다. 언급 한 바와 같이, 다른 TRP 채널뿐만 아니라 다른 STIM 및 Orai 단백질은 골격근에서 발현되며, 각각의 역할은 지금까지 결정되지 않았다. 노크 d함으로써그들의 발현 수준을 소유하고 있기 때문에 SOCE에서의 함의와 인간 골격근에서의 역할을 조사 할 수 있습니다. SOCE 측정은 전기 생리 학적 전류 기록과 Ca 2+ 측정의 두 가지 접근법을 사용하여 수행 할 수 있습니다. 전기 생리학은 확실히 현재의 관심과 그에 관련된 전기 생리 학적 신호를 측정하기 때문에보다 직접적인 방법입니다. 그러나,이 기술은 근육 세포에 적용하기가 매우 어렵습니다. 주로 근육 세포의 크기가 크고 내인성 SOCE 전류가 작기 때문입니다. 세포질 칼슘 촬상 기술적으로 매우 접근이지만, 세포질에서 측정 된 Ca2 + 수준의 칼슘의 유입 2+ 및 재 펌핑 셀로부터 또는 내부 저장에 모두 반영 같이 측정은 간접적이다. 인간의 뼈의 작은 조각으로부터 myoblasts를 분리하는 것을 포함하는 방법론효소 소화, 증폭 및 FACS 후 etal 근육이이 종이에 설명되어 있습니다. 시간이 지남에 따라 차별화 마커의 표현을 따르는 근육 차별화 및 면역 형광 프로토콜의 과정은 18 에 설명되어 있습니다. 마지막으로, SOCE의 측정과 칼슘 2 + 신호 및 골격근 분화에 다른 이온 채널의 역할이 설명되어 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Dexamethansone</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">D1756</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Insulin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">I9278</td>
			<td style="width:201px;"></td>
		</tr>
		<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>
			<td style="width:201px;"></td>
		</tr>
		<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td style="width:201px;"></td>
		</tr>
		<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>
			<td style="width:201px;"></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<tr height="25">
			<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

위성 세포 (SC)는 근섬유의 원형질 막과 주변 기저 층 사이에 위치한 근육 줄기 세포입니다. 그들은 근육 재생에 필수적입니다. 골격근에서 자주 발생하는 부상시 SC가 활성화됩니다. 그들은 근육 아세포로 증식하고 근육 병변을 치료하기 위해 분화합니다. 근육 분화 동안 일어나는 많은 사건 중에서, 세포질 Ca 2+ 신호는 매우 중요합니다. Ca 2+ 신호는 Ca 2+ 가 내부 Ca 2+ 저장소에서 방출 될뿐만 아니라 세포 외부 공간에서 칼슘 2+ 가 들어간 것, 특히 상점 운영 Ca 2+ 입구 (SOCE)에서 발생합니다. 이 논문은 정형 외과 수술 후 수집 된 근육 샘플로부터 인간 근섬유의 순수한 집단을 얻기 위해 사용 된 방법을 설명합니다. 조직은 기계적 및 효소 적으로 소화되며, 세포는 증폭되어 speci의 존재에 따라 유동 세포 계측법에 의해 분류된다fic 막 마커. 일단 얻은 인간의 myoblasts 확장 및 배양 매체에서 성장 인자를 제거하여 차별화에 전념. 특정 전사 인자 및 시험 관내 면역 형광의 발현 수준은 대조 조건 및 Ca 2+ 신호 전달에 관여하는 단백질을 정지시킨 후에 근원적 인 분화 과정을 평가하는데 사용된다. 마지막으로, Fura-2의 비율을 SOC의 신뢰성 있고 재현성있는 측정을 제공하는 비율 계 Ca 2+ 프로브로 사용합니다.

인간 근육 샘플의 효소 해리 후, 세포는 GM에서 증폭되었다. CD56 + / CD146 + / CD45- / CD34- / CD144- 세포로 정의 된 사람의 myoblasts는 FACS 후에 얻어졌다. Myoblast는 분석 된 인구의 60 % 이상을 차지했습니다 ( 그림 1 ). 일차 인간의 myoblasts는 재결합, 합류로 성장하고, DM에서 48 시간 동안 배양했다. 전사 인자 MEF2와 근육 특이 적 단백질 myosin heavy chain (MyHC)의 발현에 대한 면역 염색 후 융?...

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

여기, 우리는 성인 근육 조직에서 인간 myoblasts의 순수 인구를 얻기위한 방법을 설명합니다. 이 세포들은 생체 외 골격근 분화를 연구하고, 특히 Ca 2+ 신호 전달에 관여하는 단백질을 연구하는데 사용됩니다.

인간의 Myoblast의 분리, Myogenic 분화의 평가 및 저장 운영 칼슘 항목 측정

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

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