1. Doku Toplama: <ol><li> 7 ile 12 hafta arasında farelerin kullanın. Genellikle, en az iki hayvan kullanılır: Biri, tazminat ve floresan-eksi izotip kontrol örnekleri için gerekli FACS tek renkli kontrolleri kurmaktır. Diğer sıralama için gerçek bir örnek. </li><li> CO 2 inhalasyon fareler Euthanize ya da tercih edilen bir etik protokole göre. Fare kürk, bir kağıt havlu, yukarı bakacak, ve% 70 etanol ile sprey üzerine koyun fareler iyice bulaşmasını önlemek için. </li><li> Forseps ile karın bölgesindeki cilt kaldırın, sonra keskin bir makas ile küçük bir uzunlamasına kesilmiş. </li><li> Peel deri zıt yönlerde iki flep (yukarı ve aşağı) altında arka bacak kaslarını tamamen açığa çıkarmak için. </li><li> Tibia her iki tarafın birlikte makas ekleme ve uzatarak Vergi kas dokusu. </li><li> Kas dokusu ayıklamak için femur için de aynı işlemi tekrarlayın. </li><li> Her fare doku için bir çanak kullanarak, 60mm Petri-yemekler tüm eksize kas dokusu yerleştirin. </li><li> Tüm fareler için yukarıdaki işlemi tekrarlayın. </li></ol> 2. Into Enzimatik Sindirim Tek Hücreler Doku ayrıştırmaları: Protokol bu bölümü boyunca, steril reaktifler ve araçları kullanmak ve steril koşullar altında çalışmak. <ol start="9"><li> Her Petri-çanak, 2 kollajenaz II mL (Sigma, kedi # 6885, 500u / ml) ve 250mm CaCl 2 stokunun 20 mcL ekleyin . </li><li> Kadar kirlenmiş çıkarmak ve atmak için dikkat iki forseps (tek dişli, 2 diş Güzel Bilim Araçlar, kedi # 11.051-10, 11.154-10) ile küçük parçalar (yaklaşık 1 mm 3), kas dokusu içine Gözyaşı mümkün olduğunca olmayan kas dokusu. </li><li> Petri-yemekleri Kapak ve 30 dakika boyunca 37 santigrat derece kuluçkaya yatmaktadır. </li><li> Petri-yemekleri Al ve tam olarak mash doku parçaları 3 ml&#39;lik bir şırınga pistonu kullanabilirsiniz. Bulaşmayı önlemek için her bir örnek için bir piston kullanın. </li><li> Her Petri yemek (~ 5 ml) steril soğuk PBS (gerekli tüm doku kurtarmak için Petri-bulaşık durulama) ekleyin ve 50 ml Falcon tüp içine doku çamur transferi </li><li> Falcon tüp şiddetle Sarsıntı, PBS ile tüp doldurmak, santrifüj @ 800rpm x 5 dakika (Eppendorf tezgah üstü Model: 5810R), ve supernatant aspire (tekrar 3 defa) </li><li> 37 derece sıcaklıkta inkübe edin, her tüp ve CaCl 2 10μL (250mm): 1 ml kollajenaz D karışımı (Roche, Kedi # 11088882001, 1.5u / ml) / Dispase II (04942078001, 2.4u / ml Roche, Kedi #) ekleyin bir saat boyunca dönme ile Santigrat. </li><li> Soğuk, steril PBS, vortex ve doku homojenize iyice pipet 5-10 ml ekleyin. </li><li> PBS ile tüpler kadar doldurun ve iyice karıştırın. </li><li> Sıvı, doku kalan büyük parça süzmek için 50 ml Falcon tüp üstüne yerleştirilir 40μm hücre süzgecinden aktarın. Eşit üç 50ml Falcon tüpleri içine her bir örnek bölün. PBS, 5 dakika sonra 8 santigrat derece @ 1600rpm santrifüj tüpleri her doldurun. </li><li> Süpernatantı atın ve boyama hücreleri toplamak. </li></ol> 3. Antikor Boyama ve sıralama: Protokol bu bölümü boyunca, steril FACS ve toplama tamponlar ve steril koşullar altında çalışmak. Ticari antikorlar, eğer uygun bir biçimde ve genellikle steril olarak kabul edilir. <ol start="20"><li> Tek renk ve izotip kontrol numuneleri için, 1 ml FACS tampon örnek tekrar süspansiyon ve dokuz 1.5mL ön etiketli Eppendorf tüpleri (100 mcL her tüp) askıya hücreleri bölmek. <ol><li> Aşağıdaki tabloya göre ayarlayın tek renk boyama karışımları: DİKKAT: leke izotip kontrol antikorların nihai konsantrasyonu, onlar için kontrol birincil antikor uymalıdır . Örneğin, anti-Sca-1 1 μg/10 6 hücreleri için kullanılan, ilgili izotip aynı konsantrasyonda olması gerekir. <table border="1"><tbody><tr><td> Tüp # </td><td> Tüp Adı </td><td> Şirket </td><td> Kedi # </td><td> klon </td><td> izotip </td><td> seyreltme </td></tr><tr><td> 1 </td><td> Sigara-boya </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td> sigma </td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td> PI </td><td> sigma </td><td> P4864 </td><td></td><td></td><td> 1ug/ml </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> CD31-FITC CD45-FITC </td><td> ebioscience </td><td> 11-0311-85 </td><td> 390 </td><td> Rat IgG2a </td><td> 500 </td></tr><tr><td> House yapımı </td><td></td><td> I3 / 2 </td><td> Rat IgG2b </td><td> 500 </td></tr><tr><td> 5 </td><td> SCA1-PECY7 </td><td> ebioscience </td><td> 25-5981-82 </td><td> D7 </td><td> Rat IgG2a </td><td> 5000 </td></tr><tr><td> 6 </td><td> α-7 APC </td><td> House yapımı </td><td></td><td> R2F2 </td><td> Rat IgG2b </td><td> 1000 </td></tr></tbody></table></li><li> Izotip kontrol boyama aşağıdaki tabloyu kullanarak karışımları ayarlayın: <table border="1"><tbody><tr><td> Tüp # </td><td> Isim tüp </td><td> Hoechst </td><td> PI </td><td> CD31- FITC </td><td> CD45- FITC </td><td> Scal- PE-CY7 </td><td> a-7- APC </td><td> Rat- IgG2b- APC </td><td> Rat- IgG2a- pecy7 </td><td> Rat- IgG2a- FITC </td><td> Rat- IgG2b- FITC </td></tr><tr><td> 7 </td><td> Izo CD31 / CD45 </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td></tr><tr><td> 8 </td><td> Izo Scal- pecy7 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td></tr><tr><td> 9 </td><td> Izo α-7 APC </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td><td> - </td></tr></tbody></table></li><li> Adım 20 uygun Eppendorf tüpe pipetleyin, tek renk ve izotop kontrol boyama karışımları (genellikle 5 ila 10 mcL karışımı boyama her numune eklenir böylece antikor konsantrasyonunu ayarlamak). İyice karıştırın ve 1 saat süreyle buz üzerinde kuluçkaya yatmaktadır. </li></ol></li><li> FACS hücre sıralama için antikor kokteyli 800μL (aşağıda) ile örnek fare hücreleri Leke. İyice karıştırın ve 1 saat süreyle buz üzerinde kuluçkaya yatmaktadır. Antikor kokteyli: Hoechst, PI, CD31-FITC, CD45-FITC, SCA-1-PECY7, α-7 APC DİKKAT: partiler arasında önemli farklılıklar var, bireysel olarak her bir antikor titre hücrelerine antikor optimal oranı tespit edilmelidir . Kullanılmak üzere antikor optimal miktarı 10 6 başına hedef hücreleri antikor μgs ifade ve aynı parti antikor kullanarak sabit tutulmalıdır. Açıkçası, hedef hücrelerin sayısı deneyler arasında önemli bir değişiklik yoksa, ya da boyama hacmi hücre sayısı ile ölçekli ise, her antikor sabit bir seyreltme de kullanılabilir. </li><li> Yıkama: 9 kontrol tüpleri her FACS tampon yaklaşık 1 ml ekleyin; örnek tüp FACS tampon yaklaşık 20ml ekleyin. </li><li> 5 dakika Eppendorf tüpleri @ 3000rpm (: 5417C Eppendorf masa üstü santrifüj, Model) santrifüjleyin. Örnek tüp @ 1600rpm (: 5810R Eppendorf masa üstü santrifüj, Model) 5 dakika boyunca santrifüjleyin. </li><li> Aspire ve tüm tüpleri süpernatant atın. </li><li> FACS tampon (kontrollerin her biri için 1 ml, sıralama örnek 4ml) yeniden süspanse hücreleri, FACS tüpler &quot;hücre süzgeç kapağı ile (BD Falcon kedi 352.235, gözenek boyutu 40μm) pipetlenir hücreleri olabilir kalan kümeleri kaldırmak için sitometresinin etkileyebilir. </li><li> Tek renk denetimleri ve izotipi kontrolleri ile FACS Sıralayıcısı ayarlayın. </li><li> 3.5 mL toplama tampon (% 95,% 5 DMEM FBS) ayrı ayrı aşağıdaki tabloda göre sıralanmış hücreleri toplayın. Normalde, her iki arka bacaklarda hasat, bir tek yabani tip fare prosedürün sonunda FACS lisansüstü sıralanmış hücreleri tarafından değerlendirilecek% 95 üstünde canlılığı ile 150.000 miyojenik progenitör hücreleri veya 100.000 adipogenic progenitör hücreler, (Şekil 1 hakkında getirebilimektedir ) Yüzey boyama dayalı miyojenik ve adipogenic atalarıdır tanımı <table border="1"><tbody><tr><td> Antikor / leke </td><td> Miyojenik progenitör (MP) </td><td> Adipogenic progenitör (AP) </td></tr><tr><td> Hoechst </td><td> + </td><td> + </td></tr><tr><td> PI </td><td> - </td><td> - </td></tr><tr><td> CD31-FITC </td><td> - </td><td> - </td></tr><tr><td> CD45-FITC </td><td&gt; - </td><td> - </td></tr><tr><td> SCA1-PECY7 </td><td> - </td><td> + </td></tr><tr><td> α-7 APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4. Nakli: <ol start="28"><li> @ 1500rpm 5 dakika otoklava Eppendorf tüpleri (1,5 ml), süpernatant ve transfer hücreleri çıkarmak sıralanmış hücreler santrifüj, @ 3000rpm santrifüj 5 dakika sonra, steril PBS (serum serbest!) 500 mcL hücreleri durulayın. Steril koşullar tüm reaktifler bakımı ve doku kültürü kaputu çalışan, yaklaşık 10 6 hücre / ml PBS içinde miyojenik progenitörlerle veya Matrigel adipogenic progenitör (BD kedi # 354.234) tekrar süspansiyon haline getirin. </li><li> Miyojenik atalarıdır veya alıcı farelere adipogenic progenitör nakli. <ol><li> Isoflouran kurumunuzun politikasına göre fare uyuşturan. </li><li> Enjeksiyon bölgesi etrafında saç Tıraş, daha sonra% 70 etanol ile ovularak cilt sterilize </li><li> 3 / 10 CC insülin enjektörü ile miyojenik atalarıdır veya adipogenic atalarıdır (= 20k hücreleri) 20μL (BD kedi # 309.300) enjekte edilir. </li></ol></li><li> Uygun bir süre sonra (üç hafta iyi çalışır, ancak donör elde edilen transgenik belirteçlerinin ifade myofibers sonra bir hafta içinde nakli genellikle belirgindir), (Sigma Kedi # T04840-2, 400 mcL Avertin intraperitoneal adım 29 alıcı fareler uyuşturan 25 mg / ml), daha sonra 15 mL% 4 PFA tarafından takip 50ml PBS (10 mcM EDTA içeren), transcardially serpmek. Gerekirse bir gecede% 20 sakaroz transfer, sonra bir gecede% 4 PFA ile hedef doku, post-fix toplayın. OCT, dondurma ve cryosection Embed. </li></ol> 5. Temsilcisi Sonuçlar: MP fare iskelet kas içine nakledilen zaman, onlar hemen öncesi mevcut myofibers ile kaynaşıyor. Bu nedenle donör hücreleri genetik etiket varsa onları alınan lifler kolayca tanınabilir olacaktır. Şekil 2 nakledilen hücreler, insan alkalin phostphatase ifade histokimyasal ortaya bir örnek gösterir. AP nakledilen sonucu nakli sitenin çevreye son derece bağlıdır: subkutan bu hücreler (bkz. Şekil 2) adipositlerde ve miyofibroblastlar kökenleri vermek nakledilen zaman. Yağlı dejenerasyon 3 olusup sürece kas dahil pek çok diğer siteler, hayatta yok. <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="Şekil 1" /> Şekil 1. Iskelet kası progenitör popülasyonlarının izolasyonu için strateji Sıralama (A) Sıralama strateji: Canlı hücreler, forward scatter ve yan dağılım dayalı tespit edildi . Hoechst boyama ölü hücreleri hariç anuclear enkaz ve propidium iyodür (PI) boyama dışlamak için kullanıldı. Hematopoetik (CD45) ve endotelyal (CD31) hücreleri sıralama kapıları çıkarıldı. Α7 + Hoechst + PI-CD45 - CD31 - scal - alt kümesi tüm miyojenik atalarıdır (MP) içerir. Scal + Hoechst + PI-CD45 - CD31 - α7 - Nüfus adipogenic atalarıdır (AP) içerir. (B) Floresan-eksi-one (FMO) izotip kontrolleri leke özgüllüğü onaylayın. (C) Saflık sıralama sonra MP ve AP altkümelerini denetler. <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="Şekil 2" /> Şekil 2. MP ve AP nakli AP Temsilcisi sonuçları subkutan nakledilen (üst panel) ve MP intramüsküler (alt panel). Her iki durumda da, donör hücreleri kahverengi lekelere tarafından tespit edilen transgenik insan alkalen fosfataz ifade eden bir fare, kaynaklanmıştır.

<ol>
	<li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+stem+cells.">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li><li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li><li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors:+A+double-edged+sword+in+skeletal+muscle+regeneration.">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li><li>Sherwood, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li><li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li></ol>

Bu protokolün amacı, yüksek verim ve yüksek saflaştırılmış hücrelerinin canlılığı arasında makul bir denge. Sağlıklı hücreleri kurtarıldı olduğunu sağlanmasında en önemli adım, tahmin edilebileceği gibi, başlangıç ​​doku enzimatik ayrılma. Doku taşıma göstermek ya da görsel-işitsel bir formatta bile takdir etmek zordur, özellikle nazik olmalıdır. Bir diğer önemli faktör, prosedürün uzunluğu. Uzun bu nedenle düşük canlılığı ve engraftman verimliliği donörden alıcı hayvan gitmek için alır. PI sıralama sonra saflaştırılmış hücre örneklerinde pozitif olay sıklığı bakarak hemen cevap değildir canlılığı ile ilgili herhangi bir soru olmalı mıdır, biz clonogenicity ölçmek için bir sınırlayıcı seyreltme tahlil 3 yapılır öneririz. Hasarsız kas tipik bir türlü rutin in vitro miyojenik veya fibrotik / adipogenic koloniler başlatma yeteneğine sahip 15-20 hücreleri yaklaşık 1 verim. Milletvekilleri başlatılan kolonilerin aslında% 100 farklılaştırılmış myotubes içerirken, FAPS elde edilen koloniler, sadece yaklaşık üçte biri, düz kas aktini pozitif miyofibroblastlar ek adipositler içerir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Kollajenaz II. </td><td> Sigma-Aldrich </td><td> C-6885, </td><td> 500 u / ml </td></tr><tr><td> Kollajenaz D </td><td> Roche </td><td> 11088882001 </td><td> 1.5 u / ml </td></tr><tr><td> II çekilin </td><td> Roche </td><td> 04942078001 </td><td> 2.4 u / ml </td></tr><tr><td> hücre süzgecinden </td><td> BD </td><td> 352340 </td><td> 40 mikron </td></tr><tr><td> Tüp hücre süzgecinden </td><td> BD </td><td> 352235 </td><td> 40 mikron </td></tr><tr><td> Hoechst33342 </td><td> Sigma-Aldrich </td><td> B2261 </td><td> 2-5 ug / ml </td></tr><tr><td> CD31-FITC </td><td> Ebiosciences </td><td> 11-0311-85 </td><td> Klon 390 </td></tr><tr><td> CD45-FITC </td><td> Ev yapımı </td><td></td><td> Klon I3 / 2 </td></tr><tr><td> SCA1-PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td> Klon D7 </td></tr><tr><td> α-7 integrin APC </td><td> Ev yapımı </td><td> Rossi laboratuar ile Inquire </td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

İskelet kası, birden fazla farklı embriyonik kökenleri ve gelişim potansiyeli progenitör popülasyonlar içerir. Genellikle myofiber plazma membran ve ensheaths bu laminin zengin bazal membran arasında bir &quot;uydu hücre pozisyonu&quot; ikamet miyojenik progenitörlerle sadece miyojenik soy 1,2 kararlıyız kendini yenileyen hücreler vardır. Biz, son zamanlarda, miyofibroblastlar ve verimli bir şekilde çoğalması girerek zarar yanıt ve doku rejenerasyonu 3,4 sırasında miyojenik hücrelere trofik destek sağlar beyaz adipositler üretebilir gemi ilişkili atalarıdır ikinci sınıf bir tarif var. Belirli bir hücre popülasyonu gelişimsel ve rejeneratif potansiyelini belirlemek için en güvenilir testlerin biri izolasyon ve 5-7 nakli dayanmaktadır. Bu amaçla biz Biz bu makalede anahat enzimatik ayrışmış kas flow sitometri, protokolleri kendi temizlemesi için optimize var. Bu yöntemi kullanarak elde edilen popülasyonları miyojenik veya fibrotik / adipogenic koloni oluşturan hücreler de içerecektir: başka bir hücre tipleri, in vitro genişleyen veya in vivo teslim kalan yeteneğine sahiptir. Ancak, bu popülasyonların bir taze sıralaması altkümelerini, koloniler kurma veya alıcıların engrafting yeteneğinden yoksun diğer kirletici hücreleri içerebilir akılda tutmak gerekir moleküler analizi (örneğin QRT-PCR) için sıralama hemen sonra kullanılır.

Watch this Scientific Journal Video about Purification of Progenitors from Skeletal Muscle at JoVE.com

Purification of Progenitors from Skeletal Muscle

Enzimatik disosiasyon, fibro / adipogenic ve fare iskelet kası miyojenik atalarıdır flow sitometri ile yüzey etiketleme ve arıtma yöntemi.

İskelet Kası progenitörlerin saflaştırılması

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

purification-of-progenitors-from-skeletal-muscle

Research

JoVE Journal

Biology

18.2K Görüntüleme.  University of British Columbia. Enzimatik disosiasyon, fibro / adipogenic ve fare iskelet kası miyojenik atalarıdır flow sitometri ile yüzey etiketleme ve arıtma yöntemi.

Video: Purification of Progenitors from Skeletal Muscle

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Biyoloji

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

İskelet Kası Mitokondrial İzolasyon

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Canlı hücreler Mağaza çalışan Kalsiyum Giriş Floresans tabanlı Ölçümü: Kültür Kanseri Hücre gelen İskelet Kası Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Zebra balığı Embriyonik İskelet Kası Lazer-yaralama

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Yetişkin İskelet Kası gelen bireysel Myofibers ve Uydu Hücre İzolasyonu ve Kültür

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

İnsan İskelet Kası Kan-damar-türevli Multipotent Öncüleri İzolasyonu

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

İskelet Kası Fibroblastlarından Eksozomların İzolasyonu ve Karakterizasyonu

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Sıçandaki İskelet Kasında Fibro-Adipojenik Progenitörlerin (FAP' ler) ve Miyojenik Progenitörlerin (Milletvekilleri) Tanımlanması, İzolasyonu ve Karakterizasyonu

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Respirometrik Testler için Fare İskelet Kasından Mitokondri İzolasyonu

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.