알레산드라 다조(Alessandra d'Azzo)는 유전학 및 유전자 치료 분야의 JFC(Jewelers for Children) 기부 의장을 맡고 있습니다. 이 작업은 NIH 보조금 R01GM104981, RO1DK095169, CA021764, 멤피스의 아시시 재단, 미국 레바논 시리아 연합 자선 단체의 일부 지원을 받았습니다.

마우스에서의 모든 시술은 세인트 주드 아동 연구 병원(St. Jude Children's Research Hospital), 기관 동물 관리 및 사용 위원회(Institutional Animal Care and Use Committee) 및 미국 국립보건원(National Institutes of Health) 가이드라인에 의해 승인된 동물 프로토콜에 따라 수행되었다.
1. 용액 및 매체 준비
<ol><li>PBS 15.4mL를 20mg/mL 콜라겐분해효소 P 2.5mL(최종 농도 5mg/mL), 11U/mL dispase II 2mL(최종 농도 1.2U/mL 최종 농도) 및 100μL의 1.0MCaCl2 (최종 농도 5mM)와 혼합하여 분해 용액을 준비합니다.</li>
	<li>DMEM 440mL와 FBS 50mL(10%), 펜/연쇄상구균 5.0mL(각각 100U/mL 및 100μg/mL) 및 글루타민 보충제 5.0mL(2mM)를 혼합하여 1차 섬유아세포 배지 500mL(DMEM 완료)를 준비합니다. 0.22μm 기공 크기의 진공 필터로 배지를 필터 멸균합니다.</li>
	<li>4°C, 100,000 x g 에서 38.5mL 폴리프로필렌 튜브에 FBS를 하룻밤 동안 초원심분리하여 엑소좀이 없는 혈청을 준비하고 상층액을 새 튜브로 옮깁니다.</li>
	<li>DMEM 440mL와 엑소좀 프리 혈청 50mL(10%), 펜/연쇄상구균 5mL(100U/mL 및 100μg/mL), 글루타민 보충제 5mL(2mM)를 혼합하여 엑소좀 프리 배지 500mL를 준비합니다. 0.22μm 기공 크기의 진공 필터로 배지를 필터 멸균합니다.</li>
	<li>0.5 mL의 dH 7.4 O에 Tris 염기 6.057g을 녹이고 HCl로 pH를 7.4로 조정하여 90 M Tris-HCl (pH2)을 준비합니다. dH100O로 부피를 2 mL로 조정합니다.</li>
	<li>10 mM Tris-HCl (pH 7.4) / 1 mM Mg (Ac) 2 용액 (자당 작업 용액)을 97.9 mL 2 M Tris-HCl (pH = 7.5) 및 100 μL의 1 M Mg (Ac) 2 μL를혼합하여 준비합니다. 사용 직전에 프로테아제 억제제를 용액에 첨가하고 용액을 얼음 위에 보관하십시오.</li>
	<li>표 1에 따라 얼음 위에 자당 밀도 구배 용액을 준비합니다. 이러한 용액은 두 개의 슈크로스 그래디언트 튜브를 준비하기에 충분합니다.</li>
	<li>TCA 분말 100g에dH2O40mL를 첨가하여 100% TCA를 제조하고 완전히 녹을 때까지 혼합합니다. dH2O로 최종 부피를 100mL로 조정합니다.</li>
	<li>20mL의dH2O와 80mL의 100% 에탄올을 혼합하여 80% 에탄올을 준비합니다.</li>
	<li>1.8L의dH2O와 200mL의 10x 러닝 버퍼를 혼합하여 웨스턴 블롯 러닝 버퍼를 준비합니다.</li>
	<li>1.4L의dH2O와 200mL의 10x 전달 완충액 및 400mL의 메탄올을 혼합하여 웨스턴 블롯 전달 완충액을 제조합니다. 전송 버퍼를 4°C로 사전 냉각합니다.</li>
	<li>20dH122 O (pH 850)의 2 mL에 180g의 트리스베이스와 180g의 염화나트륨을 녹여 8.0x 트리스 완충 식염수 (TBS)를 준비합니다. dH 1O로 볼륨을2L로 조정합니다.</li>
	<li>무지방 분유 5g을 10% Tween-20 1mL, 20x TBS 5mL 및 dH2O94mL와 함께 녹여 차단 완충액을 준비합니다.</li>
	<li>BSA 3g을 10% Tween-20 1mL, 20x TBS 5mL 및 dH2O94mL와 함께 용해시켜 항체 완충액을 준비합니다.</li>
	<li>100mL의 20x TBS와 20mL의 10% Tween-20(10x)을 1.88L의dH2O와 혼합하여 세척 완충액을 준비합니다.</li>
	<li>luminol/enhance 1 부피와 안정한 과산화물 완충액 1 부피를 혼합하여 현상액을 준비합니다.</li>
</ol>2. 비복근(GA) 마우스 근육의 해부15,16
<ol><li>얼음 위에 10mL PBS가 들어 있는 50mL 튜브를 준비합니다.</li>
	<li>CO2 챔버에서 마우스를 안락사시킨 후 자궁경부 탈구를 실시합니다.</li>
	<li>양쪽 다리에서 비복근을 제거하고 얼음 위에 PBS가 있는 튜브로 옮깁니다. 알림: GA 근육을 PBS로 옮기기 전에 GA 근육에서 가자미 근육을 제거하십시오.</li>
</ol>3. 마우스 1차 섬유아세포 분리 및 배양
<ol><li>근육의 무게를 측정하고 생물 안전 후드 아래의 10cm 접시로 옮깁니다. 고운 반죽이 될 때까지 메스로 근육을 다집니다.</li>
	<li>잘게 다진 근육 페이스트를 50mL 튜브에 옮기고 3.5x 부피/mg 조직의 분해 용액을 튜브에 넣고 37°C에서 45분 동안 배양합니다. 현탁액을 10분마다 5mL 피펫으로 완전히 혼합합니다(이렇게 하면 완전한 해리에 도움이 됩니다). 알림: 또는 이 단계에 조직 해리기를 사용할 수 있습니다.</li>
	<li>세포 현탁액에 20mL의 DMEM complete를 추가하여 분해 용액을 비활성화합니다. 50mL 튜브에 놓인 70μm 나일론 셀 스트레이너로 옮깁니다. 플로우 스루를 수집하고 추가로 5mL의 DMEM complete로 세포 스트레이너를 세척합니다.</li>
	<li>실온(RT)에서 300 x g 에서 10분 동안 셀 현탁액을 원심분리하고 상층액을 조심스럽게 제거합니다.</li>
	<li>펠릿을 10mL의 DMEM 완료에 재현탁시키고 세포를 10cm 접시에 파종합니다.</li>
	<li>37 °C, 5 % CO 2, 3 % O2 (통로 0 또는 P0)에서 세포를 배양합니다. 참고: 1) 이러한 배양은 생리학적으로 유사한 조건(골격근의 O 2 수준은 약2.5%)을 보장하기 위해 낮은 산소 수준으로 유지되어야 합니다17. 2) 세포 배양의 통로 번호(예: P0)는 배양이 과대 배양된 횟수의 기록입니다. 3) P0의 일차 섬유아세포는 100% 밀도에 1일을 더할 때까지 일상적으로 배양됩니다(P0에 한함). 이는 다른 유형의 세포를 제거하고 배양에 존재하는 세포가 현미경 검사를 기반으로 근원 세포가 고갈된 섬유아세포일 뿐임을 확인하기 위한 것입니다. 생후 2개월이 된 성체 동물의 골격근에는 약 2%의 근생성 전구세포가 포함되어 있다4. 또한, myogenic progenitors는 일반적으로 코팅되지 않은 접시에 부착되지 않습니다. 그러므로, 그들은 서브컬쳐(subculturing) 18,19,20 동안에 손실된다. 근육 세포는 20% FBS와 염기성 섬유아세포 성장 인자(bFGF)가 보충된 다른 배지(F10)가 필요합니다18. DMEM complete에서 중간 섬유아세포는 근육 세포보다 증식 속도가 더 높기 때문에 첫 번째 통과 전에 이러한 오염 세포가 제거됩니다.</li>
	<li>P0 세포를 PBS로 헹구고 100%가 합류하면 1일을 더합니다. 트립신화 용액 1mL를 첨가하고 37°C, 5% CO2, 3%O2에서 배양하여 세포를 분리합니다. 10mL의 DMEM complete를 사용하여 효소 활성을 중지합니다.</li>
	<li>세포를 RT에서 10분 동안 300 x g 으로 원심분리하고 펠릿을 20mL의 DMEM 완료에 재현탁시키고 15cm 접시(통로 1 또는 P1)에 파종합니다. 세포는 80%–90% 밀도까지 성장하며 통로 3까지 확장할 수 있습니다. 참고: 다운스트림 실험 통로 1에 따라 통로 2(P2) 또는 통로 3(P3)을 사용할 수 있습니다.</li>
</ol>4. 세포의 파종 및 조건화된 배지의 수집
<ol><li>섬유아세포(P1, P2 또는 P3)를 10mL의 PBS로 세척하고 2.5mL의 트립신화 용액을 세포에 첨가하고 37°C, 5% CO2, 3%O2에서 배양합니다. 10mL의 DMEM complete를 사용하여 효소 활성을 중지합니다. 참고: 4번 통과 이후에는 1차 전지가 폐기되고 특성이 변경되므로 추가 실험에 사용해서는 안 됩니다.</li>
	<li>세포를 수집하고 상온에서 300 x g 에서 10분 동안 원심분리합니다.</li>
	<li>세포 펠릿을 10mL의 엑소좀이 없는 배지에 재현탁시키고 세포를 계수합니다.</li>
	<li>접시 당 1.5-2.0 x 106 셀 (15cm)에서 세포를 파종하고 37 °C, 5 % CO 2, 3 % O2 에서 배양합니다.</li>
	<li>얼음 위의 50mL 튜브에서 16–24시간 사이에 조절된 배지를 수집합니다. 참고: 세포가 80% 합류하지 않으면 새로운 엑소좀 유리 배지를 다시 추가하고 추가 16-24시간 후에 수집할 수 있습니다. 추가 처리를 위해 두 컬렉션을 결합할 수 있습니다.</li>
</ol>5. 차등 및 초원심분리를 이용한 엑소좀 정제
알림: 모든 단계는 4°C 또는 얼음 위에서 수행됩니다. 필요한 경우 물을 채운 튜브로 튜브의 균형을 맞춥니다.
<ol><li>살아있는 세포를 제거하기 위해 300 x g 에서 10분 동안 조절된 배지를 원심분리하고 상층액을 새 50mL 튜브로 옮깁니다.</li>
	<li>2,000 x g 에서 10분 동안 원심분리하여 죽은 세포를 제거하고 상층액을 38.5mL 폴리프로필렌 튜브로 옮깁니다.</li>
	<li>상층액을 10,000 x g 에서 30분 동안 원심분리하여 세포 기관, 세포 사멸체 및 막 조각을 제거합니다. 상층액을 38.5mL 초투명 튜브로 옮깁니다.</li>
	<li>상층액을 100,000 x g 에서 1.5시간 동안 초원심분리하여 엑소좀을 펠트합니다.</li>
	<li>상층액을 조심스럽게 버리고 약 1mL의 컨디셔닝된 배지를 남기고 엑소좀 펠릿을 총 30mL의 얼음처럼 차가운 PBS로 세척합니다.</li>
	<li>100,000 x g 에서 1.5시간 동안 원심분리하고 엑소좀 펠릿을 방해하지 않도록 주의하면서 피펫팅으로 상층액을 조심스럽게 버립니다.</li>
	<li>펠릿을 얼음처럼 차가운 PBS(15cm 접시당 ~25μL)에 재현탁시키고 BCA 단백질 분석 키트와 562nm에서 마이크로플레이트 리더를 사용하여 단백질 농도를 측정합니다. 참고: 단백질 농도는 dH2O에서 0.1% Triton-X100으로 1:2 희석하여 측정합니다. 80% 밀도에서 1차 섬유아세포의 15cm 접시(20mL의 조절된 배지)에서 엑소좀의 수율은 ~3–4μg입니다.</li>
</ol>6. 슈크로스 밀도 구배에 의한 엑소좀의 특성 분석
<ol><li>2.0 M의 1.5 mL, 1.3 M의 2.5 mL, 1.16 M의 2.5 mL, 0.8 M의 2.0 mL 및 0.5 M 슈크로스 용액의 2.0 mL를 피펫팅하여(아래에서 위로) 13 mL 초투명 튜브에 슈크로스 그래디언트를 로드합니다.</li>
	<li>30–100 μg의 엑소좀을 0.25 M 자당 용액과 혼합하고 부피를 1 mL로 조절합니다.</li>
	<li>그래디언트 위에 엑소좀을 조심스럽게 로드하고 100,000 x g 및 4°C에서 2.5시간 동안 샘플을 초원심분리합니다.</li>
	<li>울트라 원심분리기에서 튜브를 꺼내 얼음 위에 놓고 그래디언트 상단에서 시작하여 1.0mL 분획을 수집합니다. 얼음 위의 1.7mL 튜브로 옮깁니다.</li>
	<li>각 튜브에 110μL의 100% TCA를 넣고 잘 섞은 후 RT에서 10분 동안 배양합니다.</li>
	<li>10,000 x g 및 RT에서 10분 동안 원심분리하고 상층액을 조심스럽게 버립니다.</li>
	<li>펠릿을 500μL의 사전 냉각된 80% 에탄올에 재현탁시키고 샘플을 -20°C에서 10분 동안 세척하도록 둡니다.</li>
	<li>10,000 x g 및 4 °C에서 10분 동안 원심분리하고 상층액을 버립니다.</li>
	<li>펠릿을 RT에서 10-15분 동안 건조시키고 시험관 내 실험을 위해 펠릿을 PBS에 재현탁시킵니다. BCA 단백질 분석 키트를 사용하여 단백질 농도를 측정하거나 웨스턴 블롯 분석을 위해 14.5μL의 dH2O, 5μL의 4x Laemmli 완충액 및 0.5μL의 1M DTT에 펠릿을 직접 재현탁시킵니다.</li>
</ol>7. 웨스턴 블롯 분석을 통한 엑소좀 검출
<ol><li>5.7 단계에서 채취한 엑소좀 5–10 μg을 5 μL의 4x Laemmli 완충액 및 0.5 μL의 1 M DTT와 혼합한 다음 dH2O를 사용하여 최종 부피를 20 μL로 조정합니다. 6.9단계의 샘플을 포함한 모든 샘플을 98°C에서 5분 동안 가열합니다.</li>
	<li>샘플을 10% TGX stain-free gel에 넣고 제조업체의 권장 사항에 따라 단백질 ladder를 로드합니다. 참고: 관심 단백질의 분자량에 따라 겔의 백분율을 선택하십시오.</li>
	<li>로딩 염료가 겔 바닥에 올 때까지 버퍼에서 ~1시간 동안 100V에서 겔을 실행합니다. 알림: 실행 시간은 사용하는 장비 또는 젤의 종류 및 비율에 따라 다를 수 있습니다.</li>
	<li>젤을 이미지화합니다. 얼룩이 없는 겔의 이미지는 면역 블록의 로딩 컨트롤로 사용됩니다.</li>
	<li>PVDF 멤브레인을 사용하여 80V에서 1.5시간 동안 또는 4°C에서 30V에서 밤새 겔을 옮깁니다.</li>
	<li>RT에서 1시간 동안 차단 완충액의 멤브레인을 차단합니다.</li>
	<li>특이 항체 멤브레인(표 2)을 항체 완충액에서 4°C에서 밤새 교반하면서 배양합니다.</li>
	<li>세척 완충액에서 멤브레인을 세척하고 적절한 2차 항체(표 2)로 멤브레인을 교반과 함께 RT에서 1시간 동안 배양합니다.</li>
	<li>세척 버퍼에서 멤브레인을 세척하고 현상액과 카메라 기반 이미저를 사용하여 멤브레인을 이미지화합니다. 또는 X선 필름을 사용하여 멤브레인을 현상합니다.</li>
</ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+biogenesis,+biologic+function+and+clinical+potential.">Exosomes: biogenesis, biologic function and clinical potential.</a> Cell &amp; Bioscience. 9 (19), 3070-3085 (2019).</li><li>van de Vlekkert, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+exosome+release+is+the+pathogenic+pathway+linking+a+lysosomal+deficiency+to+generalized+fibrosis.">Excessive exosome release is the pathogenic pathway linking a lysosomal deficiency to generalized fibrosis.</a> Science Advances. 5 (7), 3270 (2019).</li><li>Rackov, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Vesicle-Mediated+Control+of+Cell+Function:+The+Role+of+Extracellular+Matrix+and+Microenvironment.">A Vesicle-Mediated Control of Cell Function: The Role of Extracellular Matrix and Microenvironment.</a> Frontiers in Physiology. 9, 651 (2018).</li><li>Howitt, J., Hill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+the+pathology+of+neurodegenerative+diseases.">Exosomes in the pathology of neurodegenerative diseases.</a> Journal of Biological Chemistry. 291 (52), 26589-26597 (2016).</li><li>Jing, H., Tang, S., Lin, S., Liao, M., Chen, H., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+extracellular+vesicles+in+renal+fibrosis.">The role of extracellular vesicles in renal fibrosis.</a> Cell Death and Disease. 10 (5), 367 (2019).</li><li>Asef, A., Mortaz, E., Jamaati, H., Velayati, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+role+of+extracellular+vesicles+and+exosomes+in+the+pathogenesis+of+cystic+fibrosis.">Immunologic role of extracellular vesicles and exosomes in the pathogenesis of cystic fibrosis.</a> Journal of Respiratory Diseases, Thoracic Surgery, Intensive Care and Tuberculosis. 17 (2), 66-72 (2018).</li><li>Cai, A., Cheng, X., Pan, X., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+exosomes+in+liver+physiology+and+pathology.">Emerging role of exosomes in liver physiology and pathology.</a> Hepatology Research. 47 (2), 194-203 (2017).</li><li>Machado, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+lysosomal+exocytosis+mediates+cancer+progression.">Regulated lysosomal exocytosis mediates cancer progression.</a> Science Advances. 1 (11), 1500603 (2015).</li><li>Tian, W., Liu, S., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+exosomes+in+cancer+metastasis.">Potential role of exosomes in cancer metastasis.</a> Biomed Research International. , 4649705 (2019).</li><li>Barile, L., Vassalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+Therapy+delivery+tools+and+biomarkers+of+disease.">Exosomes: Therapy delivery tools and biomarkers of disease.</a> Pharmacology and Therapeutics. 174, 63-78 (2017).</li><li>Patel, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+exosome+isolation+methods+using+culture+supernatant+for+optimum+yield,+purity+and+downstream+applications.">Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications.</a> Science Reports. 9 (1), 5335 (2019).</li><li>Ayala-Mar, S., Donoso-Quezada, J., Gallo-Villanueva, R. C., Perez-Gonzalez, V. H., Gonz&#225;lez-Valdez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+and+challenges+in+the+recovery+and+purification+of+cellular+exosomes.">Recent advances and challenges in the recovery and purification of cellular exosomes.</a> Electrophoresis. 40 (23-24), 3036-3049 (2019).</li><li>Boutagy, N. E., et al. <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+Mitochondria+from+Minimal+Quantities+of+Mouse+Skeletal+Muscle+for+High+Throughput+Microplate+Respiratory+Measurements.">Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements.</a> Journal of Visualized Experiments. (105), e53217 (2015).</li><li>. Muscle Dissection in mouse Available from: <a target="_blank" href="https://www.youtube.com/watch?v=Wh0gXfrHaH8">https://www.youtube.com/watch?v=Wh0gXfrHaH8</a> (2016)</li><li>Mas-Bargues, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relevance+of+oxygen+concentration+in+stem+cell+culture+for+regenerative+medicine.">Relevance of oxygen concentration in stem cell culture for regenerative medicine.</a> International Journal of Molecular Sciences. 20 (5), 1195 (2019).</li><li>Bois, P. R., Grosveld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FKHR+(FOX01a)+is+required+for+myotube+fusion+of+primary+mouse+myoblasts.">FKHR (FOX01a) is required for myotube fusion of primary mouse myoblasts.</a> The EMBO Journal. 22 (5), 1147-1157 (2003).</li><li>Machida, S., Spangenburg, E. E., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+rat+muscle+progenitor+cells+have+decreased+proliferation+and+myotube+formation+during+passages.">Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages.</a> Cell Proliferation. 37, 267-277 (2004).</li><li>Syverud, B. C., Lee, J. D., VanDusen, K. W., Larkin, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+satellite+cells+for+skeletal+muscle+tissue+engineering.">Isolation and purification of satellite cells for skeletal muscle tissue engineering.</a> Journal of Regenerative Medicine. 3 (2), 117 (2014).</li></ol>

저자 중 누구도 선언할 이해 상충이 없습니다.

이 프로토콜에 설명된 바와 같이 배양 배지에서 엑소좀을 성공적으로 분리하기 위한 중요한 단계는 성체 골격근에서 1차 마우스 섬유아세포 배양을 적절하게 확립하고 유지하는 것입니다. 이러한 배양은 생리학적으로 유사한 조건(골격근의 O2 수준은 ~2.5%)을 보장하기 위해 낮은 산소 수준으로 유지되어야 합니다15. 원발성 섬유아세포는 배양에 너무 많이 통과되면 형질?...

엑소좀은 크기가 30–150nm인 이질적인 세포외 소포체입니다. 그들은 생리학 및 병리학적 과정에서 확립된 핵심 플레이어이며, 조직과 장기에 유비쿼터스 분포를 가지고 있습니다 1,2. 엑소좀은 단백질, 지질, DNA 유형 및 RNA 유형의 복잡한 화물을 운반하며, 이는 파생된 세포의 유형에 따라 다릅니다 1,2,3. 엑소좀은 서로 다른 기능을 하는 단백질(즉, CD9 및 CD63을 포함한 테트라스파닌)이 풍부하여 융합 현상을 담당합니다. 예를 들어, 열 충격 단백질 HSP70 및 HSP90은 항원 결합 및 제시에 관여합니다. 또한 Alix, Tsg101 및 flotillin은 엑소좀 생물 발생 및 방출에 관여하며 이러한 나노 소포 2,3,4의 마커로 널리 사용됩니다.
엑소좀은 또한 수용 세포로 전달될 수 있는 다양한 RNA(즉, microRNA, long noncoding RNA, ribosomal RNA)를 포함하며, 이는 다운스트림 신호전달에 영향을 미칩니다3. 엑소좀 생리활성은 단일 단위막으로 둘러싸여 있기 때문에 단백질과 핵산의 화물뿐만 아니라 제한막의 지질 성분에도 의존한다1. 엑소좀 막에는 포스파티딜세린, 포스파티드산, 콜레스테롤, 스핑고미엘린, 아라키돈산 및 기타 지방산이 풍부하며, 이 모든 것이 엑소좀 안정성과 막 토폴로지에 영향을 미칠 수 있습니다 2,3. 화물과 지질 배열의 결과로 엑소좀은 수용 세포에서 신호 전달 경로를 시작하고 정상 조직 생리의 유지에 관여합니다 1,2,4,5. 특정 병리학적 조건(예: 신경 퇴행, 섬유증 및 암)에서 병리학적 자극을 유발하고 전파하는 것으로 나타났습니다 4,6,7,8,9,10,11.
엑소좀은 인접 부위나 먼 부위에 신호를 전파할 수 있는 능력으로 인해 질병 상태의 진단 또는 예후에 중요한 바이오마커가 되었습니다. 또한 엑소좀은 치료용 화합물 2,12의 매개체로 실험적으로 사용되었습니다. 클리닉에서 이러한 나노 소포의 잠재적 인 적용은 최대 수율, 순도 및 재현성을 달성하기 위해 분리 방법을 점점 더 중요하게 만듭니다. 엑소좀을 분리하기 위한 다양한 기술이 개발되고 구현되었습니다. 일반적으로 엑소좀은 차등 원심분리, 크기 배제 크로마토그래피, 면역 포획(시판 키트 사용)을 통해 조건화된 세포 배양 배지 또는 체액에서 분리할 수 있습니다. 각 접근 방식에는 이전에 논의된 고유한 장점과 단점이있습니다 1,2,13,14.
개요된 프로토콜은 1) 성체 마우스 비복근에서 일차 섬유아세포를 분리하고 배양하며, 2) 이러한 세포에 의해 배양 배지로 방출되는 엑소좀의 정제 및 특성 분석에 중점을 둡니다. 기능성 연구를 위해 원발성 섬유아세포에서 엑소좀을 분리하기 위한 잘 확립된 프로토콜은 현재 부족합니다. 일차 섬유아세포는 많은 양의 엑소좀을 분비하지 않기 때문에 분리 및 정제 과정이 까다롭습니다. 이 프로토콜은 형태학적 무결성과 기능적 활성을 유지하면서 대량의 배양물에서 다량의 순수 엑소좀을 정제하는 방법을 설명합니다. 조건화된 배지에서 얻은 정제된 엑소좀은 in vitro 흡수 실험에서 성공적으로 사용되어 수용 세포에서 특정 신호 전달 경로를 유도했습니다. 또한 여러 생물학적 샘플에서 추출한 엑소좀 화물의 비교 단백질체 분석에도 사용되어 왔다4.

<table>
	<tbody>
		<tr>
			<td>10 cm dishes</td>
			<td>Midwest Scientific, TPP</td>
			<td>TP93100</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm dishes</td>
			<td>Midwest Scientific</td>
			<td>TP93150</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo Fisher Scientific, Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Fraction V</td>
			<td>Roche</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11</td>
			<td>Eppendorf</td>
			<td>022620659/5427754008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemidoc MP imaging system</td>
			<td>BioRad</td>
			<td>12003154</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma, Roche</td>
			<td>11 213 857 001</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>cOmplete protease inhibitor cocktail</td>
			<td>Millipore/Sigma, Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion Blotter with plate electrodes</td>
			<td>BioRad</td>
			<td>1704070</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion TGX stain-free protein gel</td>
			<td>BioRad</td>
			<td>5678034</td>
			<td>10% 18-well, midi-gel</td>
		</tr>
		<tr>
			<td>Criterion vertical electrophoresis cell (midi)</td>
			<td>BioRad</td>
			<td>NC0165100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma, Roche</td>
			<td>04 942 078 001</td>
			<td>neutral protease, grade II</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma/Millipore, Roche</td>
			<td>10708984001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modification Eagles Medium</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Pharmco by Greenfield Global</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluostar Omega multi-mode microplate reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P Transfer membranes</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer (4x)</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate solution</td>
			<td>Sigma</td>
			<td>63052-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>LabScientific</td>
			<td>M-0842</td>
			<td></td>
		</tr>
		<tr>
			<td>O2/CO2 incubator</td>
			<td>Sanyo</td>
			<td>MC0-18M</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Premium microcentrifuge tubes</td>
			<td>Fisher Scientific, Midwest Scientific</td>
			<td>AVSC1510</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Protected disposable scalpels</td>
			<td>Fisher Scientific, Aspen Surgical Bard-Parker</td>
			<td>372610</td>
			<td></td>
		</tr>
		<tr>
			<td>Running buffer</td>
			<td>BioRad</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick release-GP sterile vacuum filtration system</td>
			<td>Millipore</td>
			<td>S2GPU05RE</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Sterile cell strainer (70 mm)</td>
			<td>Fisher Scientific, Fisher brand</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal west Femto</td>
			<td>Thermo Fisher Scientific</td>
			<td>34096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall Polypropylene tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>BioRad</td>
			<td>16110734</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma</td>
			<td>91228-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>BioRad</td>
			<td>1610719</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100 solution</td>
			<td>Sigma</td>
			<td>93443-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>12604-013</td>
			<td>No phenol red</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>BioRad</td>
			<td>#1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge Optima XPM</td>
			<td>Beckman Coulter</td>
			<td>A99842</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (14x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tubes (25x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Isotemp 220</td>
			<td>Fisher Scientific</td>
			<td>FS220</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of exosomes from skeletal muscle fibroblasts

엑소좀은 거의 모든 세포에서 방출되고 모든 생체액에서 분비되는 작은 세포외 소포체입니다. 이러한 소포를 분리하기 위해 초원심분리, 한외여과 및 크기 배제 크로마토그래피를 포함한 많은 방법이 개발되었습니다. 그러나 모든 엑소좀이 대규모 엑소좀 정제 및 특성화에 적합한 것은 아닙니다. 여기에는 성체 쥐 골격근에서 분리한 일차 섬유아세포의 배양을 확립한 후 이들 세포의 배양 배지에서 엑소좀을 정제하고 특성 분석하기 위한 프로토콜이 요약되어 있습니다. 이 방법은 순차적 원심분리 단계 후 자당 밀도 구배를 사용하는 것을 기반으로 합니다. 그런 다음 엑소좀 제제의 순도는 표준 마커(예: Alix, CD9 및 CD81)를 사용한 웨스턴 블롯 분석을 통해 검증됩니다. 이 프로토콜은 전자 현미경, 질량 분석법 및 기능 연구를 위한 흡수 실험을 위해 생체 활성 엑소좀을 분리하고 농축하는 방법을 설명합니다. 쉽게 확장 또는 축소할 수 있으며 다양한 세포 유형, 조직 및 생물학적 체액에서 엑소좀 분리에 적용할 수 있습니다.

이 프로토콜은 비용 효율적인 방식으로 대량의 조절된 배지에서 엑소좀을 정제하는 데 적합합니다. 이 절차는 재현성이 높고 일관성이 있습니다. 그림 1 은 생쥐 일차 섬유아세포의 배양액에서 정제된 엑소좀의 투과전자현미경(TEM) 이미지를 보여줍니다. 그림 2 는 표준 엑소좀 마커의 단백질 발현 패턴과 세포질(LDH) 및 ER(칼넥신) 단백질 오염 물질의 부...

Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

이 프로토콜은 1) 성체 마우스 비복근에서 일차 섬유아세포를 분리하고 배양하는 방법과 2) 자당 밀도 구배와 웨스턴 블롯 분석을 결합한 차등 초원심분리 방법을 사용하여 엑소좀을 정제하고 특성화하는 방법을 보여줍니다.

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 ...

isolation-characterization-exosomes-from-skeletal-muscle

Research

JoVE Journal

Biology

3.2K 조회수.  St. Jude Children's Research Hospital. 이 프로토콜은 1) 성체 마우스 비복근에서 일차 섬유아세포를 분리하고 배양하는 방법과 2) 자당 밀도 구배와 웨스턴 블롯 분석을 결합한 차등 초원심분리 방법을 사용하여 엑소좀을 정제하고 특성화하는 방법을 보여줍니다.

비디오: Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

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 ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

골격근에서 Progenitors의 정화

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

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA 함유 Exosomes의 분리 및 특성

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

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 of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

고해상도 호흡율 측정을위한 차동 원심 분리에 의한 골격근에서 본래 미토콘드리아의 분리

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

성인 심장 섬유 아세포 및 근섬유 아세포의 격리 및 특성화

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

진동절단 심근슬라이스에서 인간 심실 심근세포의 분리

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...