1. 조직 컬렉션 : <ol><li> 7 일 사이와 12 주 된 마우스를 사용합니다. 보통, 적어도 두 동물이 사용됩니다 : 하나는 보상과 형광 - 마이너스 - 원 isotype 컨트롤 샘플에 필요한 외과 단일 색상 컨트롤을 설정하는 것입니다. 다른 분류에 대한 실제 예제입니다. </li><li> CO 2 흡입하여 쥐를 안락사 또는 선택의 윤리 규정에 따라. 종이 타월, 최대 직면하고, 70 % 에탄올로 스프레이 플레이스 마우스는 철저하게 마우스 모피와 오염을 방지합니다. </li><li> 포셉와 복부의 피부를 리프트, 그리고 날카로운 가위로 작은 길이 절단합니다. </li><li> 필 반대 방향으로 피부의 두 날개는 (위, 아래)를 완전히 아래 뒷다리 사지 근육을 노출합니다. </li><li> 발목 부분의 경골의 양쪽을 따라 가위를 삽입하고 확장하여 엑사이스 근육 조직. </li><li> 근육 조직을 추출하는 대퇴골에 대해 동일한 과정을 반복합니다. </li><li> 각 마우스에서 조직 한 접시를 사용하여, 60mm 배양 - 요리의 모든 excised 근육 조직을 놓습니다. </li><li> 모든 마우스에 대해 위의 단계를 반복합니다. </li></ol> 2. 효소 소화에 의해 단일 ​​셀에 조직을 떼어 놓다 : 프로토콜의이 섹션 동안 살균 시약 및 도구를 사용하여 멸균 조건하에 작동합니다. <ol start="9"><li> 각 페트리 접시에 Collagenase - II 2 ML (시그마, 고양이 # 6885, 500u / ML) 및 250mM CaCl 2 주식 20 μL를 추가합니다. </li><li> 많은 오염을 제거하고 폐기에주의를 기울이고이 포셉 (한 톱니 모양의, 파인 과학 도구에서이 치아 한, 고양이 # 11051-10, 11154-10)과 작은 조각 (약 1 mm 3)로 근육 조직을 테어 가능한 비 근육 조직. </li><li> 페트리 - 요리를 커버하고 30 분 섭씨 37도에서 그들을 품어. </li><li> 페트리 - 요리를 검색하고 철저하게 매쉬 조직 조각 3 ML의 주사기의 플런저를 사용합니다. contaminations을 피하기 위해 각 샘플에 대해 하나 플런저를 사용합니다. </li><li> 각 페트리 - 음식 일부 (~ 5 ML) 살균 차가운 PBS를 추가하고 (필요한 모든 조직을 복구하는 경우 페트리 접시를 씻어 -) 50mL 팔콘 튜브로 조직 슬러지를 전송 </li><li> 적극적으로 팰컨 튜브 쉐이크, PBS로 튜브를 기입, 원심 분리기 @ 800rpm X 5 분 (Eppendorf 벤치 최고 모델 : 5810R), 그리고 표면에 뜨는를 기음 (반복 3 회) </li><li> 37도에서 품어, 각각의 튜브와 CaCl 2 10μL (250mM) : 1mL Collagenase D의 혼합 (로체, ​​고양이 # 11088882001, 1.5u / ML) / Dispase II (04,942,078,001, 2.4u / ML 로체, 고양이 #)을 추가 한 시간 만이라도 회전 섭씨. </li><li> 감기, 멸균 PBS, 소용돌이 그리고 조직을 균질하기 위해 철저하게 피펫의 50-10 ML을 추가합니다. </li><li> PBS로 튜브를 가득 채우고 잘 섞는다. </li><li> 조직의 나머지 큰 조각을 필터링하는 50 ML 팰컨 튜브의 상단에 위치 40μm 셀 스트레이너에 액체를 전송합니다. 고르게 세 50mL 팰컨 튜브에 각각의 샘플을 나누십시오. PBS는 다음 섭씨 8 도에 5 분 @ 1600rpm을 원심 분리기로 튜브의 각을 입력합니다. </li><li> 뜨는을 취소하고 얼룩을 위해 세포를 수집합니다. </li></ol> 3. 항체 스테 이닝과 정렬 : 프로토콜의이 섹션 동안 살균 외과 및 수집 버퍼를 사용하여 멸균 조건하에 작동합니다. 적절히 치료하면 상업 항체는 일반적으로 멸균 간주됩니다. <ol start="20"><li> 단일 색상과 isotype 컨트롤 샘플, 1mL 외과 버퍼에 샘플을 resuspend 아홉 1.5mL 미리 표시 Eppendorf 튜브 (100 μL 각 튜브)에서 일시 중지 세포를 나눕니다. <ol><li> 단일 색상의 얼룩을 설정하면 다음 표에 따라 믹스 : 주의 : 착색에 isotype 제어 항체의 최종 농도가 그들이 관리하는 주요 항체의 일치해야합니다. 안티 무서 - 1이 1 μg/10 6 세포를 사용하는 경우 예를 들어, 해당 isotype은 같은 농도에서 사용해야합니다. <table border="1"><tbody><tr><td> 튜브 # </td><td> 튜브의 이름 </td><td> 회사 </td><td> 고양이 # </td><td> 클론 </td><td> isotype </td><td> 노동 희석 </td></tr><tr><td> 1 </td><td> 비 - 얼룩 </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td> 시그마 </td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td> PI </td><td> 시그마 </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> 쥐 IgG2a </td><td> 500 </td></tr><tr><td> 하우스 만든 </td><td></td><td> I3 / 2 </td><td> 쥐 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> 쥐 IgG2a </td><td> 5000 </td></tr><tr><td> 6 </td><td> α - 7의 APC </td><td> 하우스 만든 </td><td></td><td> R2F2 </td><td> 쥐 IgG2b </td><td> 1000 </td></tr></tbody></table></li><li> isotype 제어 얼룩은 다음 테이블을 사용하여 믹스 설정 : <table border="1"><tbody><tr><td> 튜브 # </td><td> 이름 튜브 </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> 쥐 - IgG2b - APC </td><td> 쥐 - IgG2a - pecy7 </td><td> 쥐 - IgG2a - FITC </td><td> 쥐 - IgG2b - FITC </td></tr><tr><td> 7 </td><td> ISO - 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> ISO - 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> ISO - 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> 단계 20에서 해당 Eppendorf 튜브에 Pipet 단일 색상과 동위 원소 제어 얼룩 믹스를 (우리가 일반적으로 혼합 얼룩 5 사이에 10 μL 각 샘플에 추가되도록 항체의 농도를 조정합니다.) 잘 믹스 1 시간 동안 얼음에 품어. </li></ol></li><li> 외과 세포 분류에 대한 항체 칵테일의 800μL (아래)와 샘플 마우스 세포를 얼룩. 잘 믹스 1 시간 동안 얼음에 품어. 항체 칵테일 : Hoechst, PI, CD31 - FITC, CD45 - FITC, Sca1 - PECY7, α - 7 APC 주의 : 세포에 항체의 최적 비율은 큰 차이가 일괄 사이에 존재하는 수 있으므로 개별적으로, 각각의 항체를 titrating에 의해 결정되어야합니다. 사용되는 항체의 최적의 금액은 10 6 표적 세포마다 항체의 μgs 표현과 항체의 동일한 일괄 처리를 사용할 때 지속적인 보관해야합니다. 분명, 대상 세포의 숫자는 실험을 사이에 크게 변화하지 않는 경우, 또는 얼룩 볼륨은 세포의 숫자와 크기를 조정하는 경우, 각 항체의 고정 희석도 사용할 수 있습니다. </li><li> 세척 : 9 제어 튜브 각 외과 버퍼의 약 1mL에 추가하고, 샘플 튜브에 외과 버퍼의 약 20mL 추가할 수 있습니다. </li><li> 5 분 Eppendorf 튜브 @ 3000rpm (5417C Eppendorf 벤치 최고 원심 분리기, 모델)을 원심 분리기. 샘플 튜브에게 5​​ 분 @ 1600rpm (5810R Eppendorf 벤치 톱 원심 분리기, 모델)을 원심 분리기. </li><li> 기음 모든 튜브의 표면에 뜨는를 삭제. </li><li> 외과 버퍼 (컨트롤 각각에 대해 1mL, 정렬 샘플 4mL)에 Resuspend 세포는 &quot;외과 튜브 &#39;셀 스트레이너 뚜껑을 통해 (BD 팰컨 고양이 # 352235, 40μm 구멍 크기)로 피펫 세포 수 있습니다 남아있는 대단히 짧은 시간을 제거하는 cytometer에 영향을 미칩니다. </li><li> 단일 색상 컨트롤과 isotype 컨트롤과 함께 외과 분류기를 설정합니다. </li><li> 3.5 ML 수집 버퍼에서 다음 테이블 (95% DMEM 5 % FBS)에 따라 개별적으로 정렬된 세포를 수집합니다. 일반적으로, 두 뒷다리 손발을 수확하면, 하나의 야생 타입 마우스는 프로 시저의 끝 부분에 외과의 reanalyzing 정렬 세포에 의해 판단 95% 위의 생존과 150,000 myogenic 전구 세포 또는 100,000 adipogenic 전구 세포 (그림 1에 대해 얻을 수 ) myogenic 및 adipogenic progenitors의 정의는 표면 얼룩을 기반으로 <table border="1"><tbody><tr><td> 항체는 / 얼룩 </td><td> Myogenic 시조 (MP) </td><td> Adipogenic 시조 (서울 = 연합 뉴스) </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 - 7의 APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4. 이식 : <ol start="28"><li> @ 1500rpm 5 분, autoclaved Eppendorf 튜브 (1.5 ML)에 뜨는 및 전송 세포를 제거 정렬 세포를 원심 분리기, 5 분 @ 3000rpm을 원심 분리기 다음 멸균 PBS (혈청 무료!) 500 μL로 세포를 씻어. 무균 조건에서 모든 시약을 유지하고 조직 문화 후드에서 근무 약 10 6 세포 / ML에서 PBS에 myogenic progenitors, 또는 Matrigel에서 adipogenic 시조 (BD 고양이 # 354234)을 resuspend. </li><li> myogenic progenitors 또는받는 마우스로 adipogenic 시조를 이식. <ol><li> isoflouran이 기관의 정책에 따라와 마우스를 마취시키다. </li><li> 사출 지역 주변의 머리를 면도, 다음 70 % 에탄올과 마찰로 피부를 소독 </li><li> 10분의 3 CC의 인슐린 주사기를 사용하여 myogenic progenitors 또는 adipogenic progenitors (= 20K 세포)의 20μL (BD 고양이 # 309300)을 주사. </li></ol></li><li> 적절한 시간 (3 주 잘 작동하지만, 기증자 파생 형질 마커를 표현 myofibers는 이식 수술 후 일주 시간 이내 증거들이) 후, (시그마 고양이 # T04840 - 2, 400 μL Avertin의 intraperitoneal 주사로 단계 29받는 쥐를 마취시키다 25 MG / ML)은 다음 4% PFA의 15mL 다음 50mL PBS (EDTA (에틸렌 다이아 민 테트라 초산) 10 μm의를 포함) 먼저와 transcardially perfuse. 필요한 경우 하루 20 %의 자당로 전송 후, 밤새 4% PFA와 대상 조직, 사후 수정 사항을 수집합니다. OCT, 동결 및 cryosection에 삽입할 수 있습니다. </li></ol> 5. 대표 결과 : MP가 마우스 골격근에 이식하는 경우, 그들은 쉽게 기존 myofibers와 퓨즈. 따라서 기증자의 세포에 존재하는 유전자 레이블을받은 섬유에서 쉽게 감지됩니다. 그림 2는 이식 세포, 인간의 알칼리성 phostphatase을 표현 histochemically 발표 예제를 보여줍니다. AP가 이식 때 결과는 이식 사이트의 환경에 크게 의존 : subcutaneously 이러한 세포 (그림 2 참조) adipocytes와 myofibroblasts에 기원을 제공 이식 때. 지방 변성은 3 유도되지 않는 근육을 포함하여 많은 다른 사이트에서는, 그들은 생존하지 않습니다. <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="그림 1" /> 그림 1. 골격근의 시조 인구의 격리를위한 전략을 정렬 (A) 정렬 전략 :. 가능한 세포는 앞으로 산란 및 측면 산란을 기반으로 확인되었습니다. Hoechst의 얼룩은 죽은 세포를 제외 anuclear 파편과 propidium 요오드화물의 (PI) 얼룩을 제외하는 데 사용되었다. 조혈 (CD45) 및 내피 (CD31) 세포가 정렬 성문에서 제외되었습니다. α7 + Hoechst + PI - CD45 - CD31 - Scal - 집합은 모든 myogenic progenitors (MP)가 포함되어 있습니다. Scal + Hoechst + PI - CD45 - CD31 - α7 - 인구 adipogenic progenitors (AP)가 포함되어 있습니다. (B) 형광 - 마이너스 - 원 (FMO) isotype 컨트롤은 얼룩의 특이성을 확인합니다. (C) 순도가 정렬 후 MP와 AP 하위 집합을 확인합니다. <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="그림 2" /> 그림 2. MP와 AP 이식. AP 다음과 같은 대표적인 결과는 subcutaneously 이식 (맨 위 패널)와 MP intramuscularly (하단 패널)되었습니다. 두 경우 모두, 기증자 세포는 갈색 얼룩으로 식별 유전자 변형 인간의 알칼리성 인산 가수 분해 효소를 표현 마우스에서 유래.

<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>

관심 없음 충돌 선언하지 않습니다.

이 프로토콜의 목적은 높은 수율과 정화 ​​세포의 높은 생존 능력 사이에 적절한 균형을 공격하는 것입니다. 건강한 세포가 회복되는 보장에서 가장 중요한 단계는 예측할 수, 시작 조직의 효소 분해됩니다. 조직의 처리 증명 또는 시청각 형식도 감사 힘든되는, 특히 부드럽게해야합니다. 또 다른 주요 요인은 절차의 길이입니다. 더 이상 그것은 따라서 낮은 생존하고, engraftment 효율, 기증자로부터받는 동물로 이동 걸립니다. 즉시 정렬 후 정화 세포 샘플에 PI 긍정적인 사건의 빈도를보고하여 답변을하지 않습니다 생존에 대한 질문이있을 경우, 우리는 clonogenicity을 측정하기 위해 제한 희석 분석은 3 수행하는 것이 좋습니다. 손상되지 않은 근육의 일반적인 종류는 정기적으로 체외에 myogenic 또는 fibro / adipogenic 식민지를 시작 할 수 15-20 세포의 약 1를 얻을 수 있습니다. 의원에서 시작한 식민지의 기본적 100 % 차별화된 myotubes가 포함되어 있지만, FAPs에서 얻은 식민지 중 고작 1 ~ 세번째는 부드러운 근육 굴지 긍정 myofibroblasts 이외에 adipocytes가 포함되어 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> Collagenase II </td><td> 시그마 - 알드리치 </td><td> C - 6885, </td><td> 500 U / ML </td></tr><tr><td> Collagenase D </td><td> 로체 </td><td> 11088882001 </td><td> 1.5 U / ML </td></tr><tr><td> II를 치환 </td><td> 로체 </td><td> 04942078001 </td><td> 2.4 U / ML </td></tr><tr><td> 셀 스트레이너 </td><td> BD </td><td> 352340 </td><td> 40 μm의 </td></tr><tr><td> 셀 스트레이너와 관 </td><td> BD </td><td> 352235 </td><td> 40 μm의 </td></tr><tr><td> Hoechst33342 </td><td> 시그마 - 알드리치 </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> 클론 390 </td></tr><tr><td> CD45 - FITC </td><td> 홈 만든 </td><td></td><td> 클론 I3 / 2 </td></tr><tr><td> Sca1 - PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td> 클론 D7 </td></tr><tr><td> α - 7 인테 APC </td><td> 홈 만든 </td><td> 로씨 연구실에 문의 </td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

골격 근육은 서로 다른 배아 기원과 발달 잠재적 여러 전구 인구가 포함되어 있습니다. 일반적으로 myofiber 플라즈마 막과 ensheaths를 laminin 풍부한 지하 막 사이에 &#39;위성 휴대폰 위치 &quot;에 거주 Myogenic progenitors은, 전적으로 myogenic 혈통 1,2하기 위해 최선을 다하고 있습니다 자기 갱신 세포 수 있습니다. 우리는 최근 myofibroblasts 효율적 확산을 입력하여 손상에 대응하고 조직 재생 3,4 중 myogenic 세포에 영양 지원을 제공하는 백색 adipocytes을 생성할 수 있습니다 선박 관련 progenitors의 두 번째 클래스를 설명합니다. 주어진 세포 인구의 발달과 재생 가능성을 결정하는 가장 신뢰할 수있는 assays 중 하나는 그들의 고립과 5-7 이식에 의존합니다. 이를 위해 우리는이 문서에서 개요 것입니다 효소 dissociated 근육에서 유동세포계측법에 의해 자신의 정화를 위해 프로토콜을 최적화합니다. 이 방법을 사용하여 얻은 인구는 myogenic 또는 fibro / adipogenic 콜로니 형성 세포 중 하나를 포함합니다 : 다른 세포 유형은 체외에서 확장 또는 생체내 전달에서 살아남기 수 없습니다. 그러나 이러한 인구가 하나가 신선한 정렬 집합이 식민지를 형성하거나 수신자를 engrafting의 능력이 결여되어 다른 오염 물질 세포를 포함할 수 있습니다 점을 염두에 보관해야 분자 분석 (예 : qRT - PCR)에 대한 정렬 즉시 사용할 때.

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

Purification of Progenitors from Skeletal Muscle

효소 분리, fibro / adipogenic 및 murine 골격근에서 myogenic progenitors의 유동세포계측법에 의해 표면 라벨 및 정화를위한 방법.

골격근에서 Progenitors의 정화

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

purification-of-progenitors-from-skeletal-muscle

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Biology

18.1K 조회수.  University of British Columbia. 효소 분리, fibro / adipogenic 및 murine 골격근에서 myogenic progenitors의 유동세포계측법에 의해 표면 라벨 및 정화를위한 방법.

비디오: 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.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

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

연구

생물학

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.

골격근에서 Mitochondrial 절연

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.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

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.

Zebrafish 배아 골격근의 레이저 쏜 부상

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.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

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.

인간의 골격 근육에서 혈관 유래 다 능성 전구체의 분리

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

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아의 분리

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

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.

골격근 섬유아세포(Skeletal Muscle Fibroblast)에서 엑소좀의 분리 및 특성 분석(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 ...

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.

쥐의 골격 근육에서 섬유 선화 (FAP) 및 근생 전구자 (의원)의 식별, 격리 및 특성화

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.

호흡 측정을 위한 마우스 골격근으로부터 미토콘드리아의 분리

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 ...