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

Detta arbete stöddes av den schweiziska National Science Foundation (bidrag nr 310030-166313), &quot;Fondation Suisse pour la recherche sur les maladies musculaires&quot;, &quot;Stiftelsen Marcel Levaillant&quot; och &quot;Fondation pour la recherche ostéo-articulaire.&quot;

Humana muskelprover (vävnader erhållna från semitendinösa muskler) samlades under ortopedisk operation på friska patienter som kirurgiskt avfall. Alla metoder relaterade till den mänskliga studien utfördes i enlighet med de schweiziska regulatoriska hälsomyndigheternas riktlinjer och föreskrifter och godkändes av Cantonale d&#39;Etique de la Recherche från Genèves kantonautoriteter, Schweiz (protokoll CER nr 12-259) . Informerade och skriftliga samtycke erhölls från alla vuxna personer som var involverade ...

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Isoleringen och kulturen av humana myoblaster från vuxen skelettmuskel erbjuder en in vitro- modell för att studera muskeldifferentiering och muskelregenerering. I det här dokumentet tillhandahåller vi ett protokoll som möjliggör rening av höga utbyten av humana myoblaster på ett enkelt och kostnadsbegränsat sätt. Dessutom ger denna teknik pålitliga och reproducerbara resultat i termer av procentuell andel av myoblaster isolerade och deras myogena differentieringseffe...

Mänskliga skelettmuskler består av grupper av kontraktil multinukleerade muskelfibrer som härrör från fusionen av myogena prekursorceller. Skelettmusklerna har kapacitet att regenerera efter skada tack vare förekomst av SC, skelettmuskelstamcellerna som ligger mellan plasmamembranet av myofibrer (sarcolemma) och basallamina. I oskadad muskel är SCs mestadels närvarande i ett vilande tillstånd. Som svar på mekanisk stress eller skada aktiveras SCs (myoblaster), prolifereras och genomgår antingen differentiering för att bilda nya myofibrer eller självförnyelse för att fylla i SC-poolen 1 , 2 . Under åren har flera tekniker utvecklats för att isolera SC och deras avkomma, myoblasterna, från skelettmuskelbiopsier. Med den större förståelsen av cellytmarkörerna uttryckta på dessa celler och metoden för fluorescensaktiverad cellsortering (FACS) 3, 4 , 5 , är det nu möjligt att isolera rena populationer av humana myoblaster från muskelprover. Kalciumsignaleringen reglerar utveckling av skelettmuskler, homeostas och regenerering. I synnerhet är Ca 2 + -posten som aktiveras efter intracellulär lagring, kallad SOCE, av stor betydelse 6 för dessa processer. Vid cellstimulering minskar Ca2 + -nivån i endo / sarkoplasmisk retikulum (ER / SR), som i sin tur aktiverar plasmametall Ca 2+ -kanaler som medger Ca 2+ -tillträde för att fylla på ER / SR 7 . De två huvudproteinerna som är involverade i SOCE är ER / SR Ca2 + -sensingstromal-interaktionsmolekylen 1 (STIM1) -proteinet och plasmamembranet Ca2 + -kanalen Orai1. Skelettmuskel uttrycker i hög grad dessa två proteiner, liksom andra proteiner av samma familjer (STIM2 aNd Orai2-Orai3) 8 , 9 och flera Ca 2+ -permeabla kanaler i den transienta receptorpotentialkanoniska (TRPC) -familjen 10 , 11 , 12 , 13 . Ca 2+ inträde genom SOCE-vägen är av stor betydelse för muskelbildning / regenerering 14 . Mutationer av STIM1- eller Orai1-proteiner har skadliga effekter på muskelfunktionen, vilket främst leder till muskelhypotoni 15 . Djurmodeller med SOCE-försämring visar också minskad muskelmassa och kraft tillsammans med ökad utmattning 6 , 16 , 17 . Som nämnts uttrycks andra STIM- och Orai-proteiner, liksom många TRPC-kanaler, i skelettmuskler, och deras respektive roller har inte hittills bestämts. Genom att knacka dÄger sin uttrycksnivå, är det således möjligt att undersöka deras konsekvenser i SOCE och deras roller under human skelettmuskeldifferentiering. SOCE-mätning kan utföras med två olika metoder: elektrofysiologiska ströminspelningar och Ca 2+ mätningar. Elektrofysiologi är verkligen en mer direkt metod, eftersom den mäter aktuell ström och dess associerade elektrofysiologiska signatur. Emellertid är denna teknik mycket svår att applicera på muskelceller, främst på grund av muskelcellernas stora storlek och den lilla storleken hos den endogena SOCE-strömmen. Cytosolisk Ca 2+ bildbehandling är tekniskt mycket tillgänglig, men åtgärden är mer indirekt, eftersom Ca 2+ -nivån uppmätt i cytosolen återspeglar både inmatningen av Ca 2+ och repumpningen ur cellen eller i de interna butikerna. En metod som inkluderar isolering av myoblaster från små bitar av mänsklig skelEtalmuskel efter enzymatisk uppslutning, förstärkning och FACS förklaras i detta dokument. Processen med muskeldifferentiering och immunofluorescensprotokollet för att följa uttrycket av differentieringsmarkörer över tiden beskrivs 18 . Slutligen förklaras mätningen av SOCE och rollen av olika jonkanaler i Ca 2 + -signalerings- och skelettmuskeldifferentiering.

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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>
		</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>
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		<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>
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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>
			<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

Satellitceller (SC) är muskelstamceller placerade mellan plasmamembranet i muskelfibrer och den omgivande basala lamina. De är viktiga för muskelregenerering. Vid skada, som uppträder ofta i skelettmuskler, aktiveras SC. De spridas som myoblaster och differentierar för att reparera muskelskador. Bland många händelser som sker under muskeldifferentiering är cytosoliska Ca 2+ signaler av stor betydelse. Dessa Ca2 + -signaler härrör från Ca2 + -frisättning från interna Ca2 + -butiker, såväl som från Ca2 + -inträde från det extracellulära utrymmet, i synnerhet den affärsdrivna Ca 2 + -inmatningen (SOCE). I detta dokument beskrivs en metod som används för att erhålla en ren population av humana myoblaster från muskelprover som samlats in efter ortopedisk kirurgi. Vävnaden smälts mekaniskt och enzymatiskt, och cellerna amplifieras och sorteras därefter genom flödescytometri beroende på närvaron av speciFic membranmarkörer. När de erhållits, expanderas humana myoblaster och engagerar sig för att differentiera genom att avlägsna tillväxtfaktorer från odlingsmediet. Expressionsnivåerna för specifika transkriptionsfaktorer och in vitro immunofluorescens används för att bedöma den myogena differentieringsprocessen i kontrollbetingelser och efter silkning av proteiner involverade i Ca 2 + -signalering. Slutligen beskriver vi användningen av Fura-2 som en ratiometrisk Ca 2+ -prov som ger pålitliga och reproducerbara mätningar av SOCE.

Efter den enzymatiska dissociationen av ett humant muskelprov förstärktes cellerna i GM. Humana myoblaster, definierade som CD56 + / CD146 + / CD45- / CD34- / CD144-celler, erhölls efter FACS. Myoblaster representerade mer än 60% av den analyserade populationen ( Figur 1 ). Primära humana myoblaster replierades, odlades till sammanflöde och odlades i DM under 48 timmar. Efter immunostaining för expressionen av transkriptionsfaktorn MEF2 och den muskelspecifika proteinet myosin tunga ...

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

Här beskriver vi en metod för att erhålla en ren population av humana myoblaster från vuxen muskelvävnad. Dessa celler används för att studera in vitro- skelettmuskeldifferentiering och i synnerhet att studera proteiner som är involverade i Ca 2 + -signaleringen.

Isolering av humana myoblaster, bedömning av myogen differentiering och butiksdriven kalciuminmatning

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.

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