Name: isolation of type i and type ii pericytes from mouse skeletal muscles
Uploaded: 2017-05-26T15:00:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

이 연구는 근력 근이영양증 재단 (MDF-FF-2014-0013)의 기금 지원과 미국 심장 협회 (16SDG29320001)의 과학자 개발 보조금으로 부분적으로 지원되었습니다.

Wildtype과 Nestin-GFP 트랜스 제닉 마우스를 미네소타 대학의 동물 시설에 넣었다. 모든 실험 절차는 미네소타 대학의 동물 동물 관리 및 사용위원회 (Institutional Animal Care and Use Committee)에 의해 승인되었으며 실험실 동물의 관리 및 사용에 관한 NIH 지침에 따라 수행되었습니다. 1. 근육 해부 및 단세포 격리 <ol><li> 트 리브로 모에 타놀 (250 mg / kg, ip)으로 성인 생쥐 (남성과 여성 모두 6-10 주)를 안락사시키고 70 % 에탄올로 복부 피부를 살균하십시오. 참고 : ketamine이 NMDA 수용체와 상호 작용하는 것으로 알려져 있기 때문에 마비 / 안락사를 위해 트라이 브로 모에탄올을 케타민 대신 여기에 사용했습니다. 이는 잠재적으로 연구에 영향을 미칠 수 있습니다. </li><li> 쥐를 앙와위에 놓고 메스를 사용하여 복부 피부에 수평 절개를하십시오. 뒷다리 근육을 드러내려면 반대 방향으로 당겨 피부를 손으로 떼어냅니다. </li><li> 콜렉포셉과 가위를 사용하여 두 개의 뒷다리에서 근육. 얼음에 1 % 페니실린 - 스트렙토 마이신 (P / S)을 보충 한 무균 인산 완충 식염수 (PBS)에 보관하십시오. </li><li> 1 % P / S 2 회 보충 된 얼음처럼 차가운 PBS에서 해부 된 뒷다리 근육을 씻고 멸균 10cm 플레이트로 옮깁니다. </li><li> 조심스럽게 2X 배율로 해부 현미경하에 포셉과 가위를 사용하여 근육, 혈관 및 결합 조직을 절개하십시오. </li><li> 가늘게 썰어 근육을 작은 조각 (1 ~ 2mm 3 )으로 멸균 가위와 칼을 사용하여 깎습니다. 필요한 경우, 근육이 건조되지 않도록하기 위해 DMEM (Dulbecco &#39;s Modified Eagle Medium)을 소량 첨가하십시오. </li><li> 기계적으로 10 ML 혈청 피펫을 통해 10 배 위아래로 pipetting하여 조직을 분해. </li><li> 혼합물에 새로 만든 소화 용액 (0.2 % 타입 2 collagenase가 보충 된 DMEM)을 첨가하십시오. 37 ° C에서 2 시간 동안 배양하십시오.35 회전 / 분에서 교반. </li><li> 혼합물을 균질화하기 위해 18 G 바늘을 사용하여 분쇄하십시오. 그런 다음 500 xg에서 5 분간 원심 분리합니다. 뜨는을 버리고 0.25 % 트립신 / EDTA에 펠렛을 resuspend. 37에서 품어 ° C 10 분. 원심 분리 단계를 두 번 더 반복합니다. </li><li> 20 % 태아 소 혈청 (FBS)을 보충 한 DMEM 10 mL를 넣고 500 xg에서 5 분간 원심 분리한다. 상등액을 버리고 적혈구 용해 완충액 (155 MM NH 4 Cl, 10 MM KHCO 3 , 0.1 MM EDTA)에 펠렛을 resuspend. </li><li> 5 분 500 XG에서 원심 분리기. 뜨는을 버리고 정렬 버퍼 (20 MM HEPES, 산도 7.0, 1 MM EDTA, 그리고 칼슘 / Mg 2 + 무료 PBS, 산도 7.0에서 1 % BSA)에 펠렛을 resuspend. 혼합물을 40 μm 셀 여과기로 여과하여 단일 세포 현탁액을 얻습니다. </li><li> 5 분 500 XG에서 원심 분리기. 뜨는을 버리고 정렬 버퍼 1 ML에 펠렛을 resuspend. </li><li> 카운트hemocytometer로 세포 수를 계산하고 정렬 버퍼에서 5 x 10 6 / mL로 단일 세포 현탁액을 희석. </li></ol> 2. 세포 염색 및 정렬 <ol><li> 표 1 에 설명 된대로 컨트롤과 샘플을 준비합니다. 표 1 에 설명 된대로 30 분 동안 얼음에 각 항체와 단일 세포 현탁액을 얼룩. </li><li> 5 분 500 XG에서 원심 분리기와 정렬 버퍼와 두 번 펠렛을 씻으십시오. </li><li> 표 1에 표시된대로 단일 세포 솔루션에 DAPI를 추가합니다. PDGFRβ-PE-FMO 컨트롤 및 샘플에 대해 DAPI 단색 컨트롤에는 5 μg / mL DAPI (최종 농도)를 사용하고 DAPI 단일 색상 컨트롤에는 1 μg / mL DAPI를 사용합니다. 실험을하는 동안 모든 튜브를 얼음에 붙이십시오. </li><li> 분류기와 소프트웨어를 켜십시오. 메시지가 나타나면 100 μm 정렬 칩을 스캔하고 삽입하십시오. </li><li> 자동 설정 ( 예 : 칩 정렬, 물방울 교정, 사이드 스트림 교정 및 정렬 지연 교정자동 설치 구슬을 넣으십시오. </li><li> 자동 설정이 완료되면 &#39;실험&#39;탭으로 이동하여 &#39;새로 만들기&#39;를 클릭하고 &#39;공개 템플릿&#39;에서 &#39;빈 템플릿&#39;을 선택하십시오. </li><li> &quot;Measurement Settings&quot;에서 &quot;FL1&quot;에 대해서는 &quot;DAPI&quot;를, &quot;FL2&quot;에 대해서는 &quot;Nestin-GFP&quot;를, &quot;FL3&quot;에 대해서는 &quot;PDGFRβ&quot;를 입력하십시오. &quot;FL4&quot;- &quot;FL6&quot;의 확인란을 선택 취소하십시오. </li><li> 확인란을 선택하여 405, 488 및 561 레이저를 활성화하고 &quot;새 실험 만들기&quot;를 클릭하십시오. </li><li> &quot;보상 마법사 시작&quot;옵션을 선택하고 &quot;보상 마법사&quot;소프트웨어 프롬프트에 따라 보상을 설정하십시오. <ol><li> 염색되지 않은 컨트롤을로드하고 &quot;시작&quot;을 클릭하십시오. &quot;감지기 및 임계 값 설정&quot;을 클릭하고 FSC 및 BSC 감지기의 센서 이득을 조정하여 인구를 저울에 배치하십시오. </li><li> 광고히스토그램의 왼쪽에 음의 모집단을 배치하는 FL1-FL3 형광 채널의 게인 레벨. &quot;기록&quot;버튼을 클릭하여 데이터를 기록하십시오. </li><li> 메시지가 표시되면 단일 색상 컨트롤을 하나씩로드하십시오. &quot;시작 및 기록&quot;을 클릭하여 데이터를 기록하십시오. 히스토그램에서 양성 집단에 대한 게이트를 조정하십시오. &quot;다음&quot;을 클릭하십시오. </li><li> &quot;보상&quot;탭에서 &quot;매트릭스 계산&quot;으로 이동 한 다음 &quot;보상 설정 계산&quot;패널에서 &quot;계산&quot;을 클릭하여 보상을 수행하십시오. &quot;마침&quot;을 클릭하여 &quot;보상 마법사&quot;를 종료하십시오. </li></ol></li><li> PDGFRβ-PE-FMO 컨트롤을로드하고 &quot;시작&quot;을 클릭하십시오. &quot;모든 이벤트&quot;플롯 아래 관심있는 세포 주위에 다각형 게이트 (게이트 A)를 그립니다. </li><li> Gate A 내부를 두 번 클릭하여 하위 플롯을 만듭니다. Y 축을 DAPI로 변경하고 라이브 (DAPI 낮음 ) 셀 주위에 다각형 게이트 (게이트 B)를 그립니다. i를 두 번 클릭하십시오.자식 플롯을 만들려면 게이트 B 옆에 있습니다. X 축을 FSC-H로 변경하고 Y 축을 FSC-W로 변경하고 이중 선을 제거하기 위해 단일 선 둘레에 다각형 게이트 (게이트 C)를 그립니다. </li><li> Gate C 내부를 두 번 클릭하여 하위 플롯을 만듭니다. X 축을 Nestin-GFP로, Y 축을 PDGFRβ-PE로 변경하십시오. &quot;기록&quot;버튼을 클릭하여 데이터를 기록하십시오. </li><li> 샘플을로드하고 2.11-2.12 단계를 반복하십시오. 녹음 후 &quot;일시 중지&quot;를 클릭하여 샘플을 보존하십시오. </li><li> PDGFRβ-PE-FMO 컨트롤을 기반으로 PDGFRβ + 및 Nestin-GFP + 세포에 대한 게이팅 경계를 정의하십시오. PDGFRβ + Nestin-GFP 및 PDGFRβ + Nestin-GFP + 개체군을위한 게이트를 그립니다. </li><li> &quot;Sorting Method&quot;에서 &quot;Two-way Tubes&quot;를 선택하고 &quot;PDGFRβ + Nestin-GFP - &quot;및 &quot;PDGFRβ + Nestin-GFP + &quot;세포를 왼쪽 및 오른쪽 수집 튜브에 할당하십시오.광적으로. 수집 버퍼에 채워진 15 mL 수집 튜브를 수집 스테이지에 놓고 &quot;Load Collection&quot;버튼을 클릭하십시오. </li><li> 샘플을 계속 실행하려면 &quot;재시작&quot;버튼을 클릭하십시오. PDGFRβ + Nestin - GFP - (유형 I pericytes)와 PDGFRβ + Nestin - GFP + (유형 II pericytes) 세포를 수집하려면 &quot;정렬 시작&quot;을 클릭하십시오. </li></ol> 3. 포스트 정렬 분석 <ol><li> 5 분 500 XG에 정렬 된 세포를 원심 분리기, pericyte 매체 ( 물질 표 참조) 1 ML에 펠렛을 resuspend, hemocytometer를 사용하여 세포 밀도를 계산합니다. </li><li> ~ 1 x 104 세포 / cm2에서 poly-D-lysine (PDL) 코팅 된 coverslips에 종자 타입 I과 타입 II pericytes. 3 % pericyte medium에서 37 ℃, 5 % CO2로 성장시킨다. </li><li> 3 일째, perisste 형태 (위상차 하에서)와 내인성 Nestin-GFP 발현을 형광 마이크로범위 (여기 레이저 : 488 nm, 여기 필터 : 470/40 nm, 방출 필터 : 515/30 nm). 20X 대물 렌즈 (0.45 NA)로 이미지를 촬영하십시오. </li><li> 지방 세포를 지방 세포 (마우스 MSC 기초 매체 + 지방 생성 자극 보조제) 및 근육 형성 (DMEM + 2 % 말 혈청) 매체로 대체하여 이전에 설명한 바와 같이 지방 형성 및 근원 분화를 각각 개시한다. 매 2-3 일마다 매체를 교체하십시오. </li><li> 상온에서 20 분 동안 4 % 파라 포름 알데히드 (PFA)에서 17 일 (지방 형성 / 근원 분화 후 14 일)에 세포를 고정시켰다. 참고 :주의 사항 : PFA는 발암 물질입니다. </li><li> 이전의 발행물 19 , 20 에 설명 된대로 perilipin (지방 세포 마커) 및 S - myosin (성숙한 myotube / myofiber 마커)에 대한 면역 세포 화학 검사를 수행합니다. <ol><li> 상온에서 10 분 동안 고정 된 세포를 PBS로 3 번 씻으십시오. </li><li> 블로킹 완충액 (PBS5 % 당나귀 혈청, 3 % BSA 및 0.3 % Triton X-100으로 보충)를 첨가하고 실온에서 1 시간 동안 배양 하였다. </li><li> 안티 perilipin (2 μg / ML) 및 / 또는 안티 S - myosin (2 μg / ML) 항체 4 ° C 하룻밤 세포를 품어. </li><li> 실온에서 10 분 동안 PBS로 세포를 3 번 ​​씻으십시오. </li><li> 1 시간 동안 실온에서 알렉사 555 당나귀 안티 토끼 (4 μg / ML) 및 / 또는 Alexa555 당나귀 안티 마우스 (4 μg / ML) 항체와 세포를 품어. </li><li> 실온에서 10 분 동안 PBS로 세포를 3 번 ​​씻으십시오. </li><li> DAPI를 포함하는 장착 매체로 면역 염색 세포를 장착하십시오 (물질 표 참조). 형광 현미경 (여기 레이저 : 543 nm, 540 / 45 nm excitation과 600/50 nm emission)을 사용하여 perilipin과 S-myosin 발현을 검사하고 40X 대물 렌즈 (0.60 NA)에서 이미지를 촬영하십시오. </li></ol></li></ol>

<ol>
	<li>Rennert, R. C., Sorkin, M., Garg, R. K., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+recruitment+after+injury:+lessons+for+regenerative+medicine.">Stem cell recruitment after injury: lessons for regenerative medicine.</a> Regenerative medicine. 7 (6), 833-850 (2012).</li><li>Sambasivan, R., 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=Pax7-expressing+satellite+cells+are+indispensable+for+adult+skeletal+muscle+regeneration.">Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration.</a> Development. 138 (17), 3647-3656 (2011).</li><li>Relaix, F., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+are+essential+for+skeletal+muscle+regeneration:+the+cell+on+the+edge+returns+centre+stage.">Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage.</a> Development. 139 (16), 2845-2856 (2012).</li><li>von Maltzahn, J., Jones, A. E., Parks, R. J., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7+is+critical+for+the+normal+function+of+satellite+cells+in+adult+skeletal+muscle.">Pax7 is critical for the normal function of satellite cells in adult skeletal muscle.</a> Proc Natl Acad Sci U S A. 110 (41), 16474-16479 (2013).</li><li>Lepper, C., Partridge, T. A., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+absolute+requirement+for+Pax7-positive+satellite+cells+in+acute+injury-induced+skeletal+muscle+regeneration.">An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.</a> Development. 138 (17), 3639-3646 (2011).</li><li>Kuang, S., Charge, S. B., Seale, P., Huh, M., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+Pax7+and+Pax3+in+adult+regenerative+myogenesis.">Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis.</a> J Cell Biol. 172 (1), 103-113 (2006).</li><li>Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A., Kardon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells,+connective+tissue+fibroblasts+and+their+interactions+are+crucial+for+muscle+regeneration.">Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration.</a> Development. 138 (17), 3625-3637 (2011).</li><li>Morgan, J. E., Pagel, C. N., Sherratt, T., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+persistence+and+migration+of+myogenic+cells+injected+into+pre-irradiated+muscles+of+mdx+mice.">Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice.</a> J Neurol Sci. 115 (2), 191-200 (1993).</li><li>Beauchamp, J. R., Morgan, J. E., Pagel, C. N., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+myoblast+transplantation+reveal+a+discrete+minority+of+precursors+with+stem+cell-like+properties+as+the+myogenic+source.">Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.</a> J Cell Biol. 144 (6), 1113-1122 (1999).</li><li>Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invited+review:+myoblast+transfer:+a+possible+therapy+for+inherited+myopathies.">Invited review: myoblast transfer: a possible therapy for inherited myopathies.</a> Muscle Nerve. 14 (3), 197-212 (1991).</li><li>Montarras, 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=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309 (5743), 2064-2067 (2005).</li><li>Asakura, A., Seale, P., Girgis-Gabardo, A., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+specification+of+side+population+cells+in+skeletal+muscle.">Myogenic specification of side population cells in skeletal muscle.</a> J Cell Biol. 159 (1), 123-134 (2002).</li><li>Tamaki, T., 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=Skeletal+muscle-derived+CD34+/45-+and+CD34-/45-+stem+cells+are+situated+hierarchically+upstream+of+Pax7++cells.">Skeletal muscle-derived CD34+/45- and CD34-/45- stem cells are situated hierarchically upstream of Pax7+ cells.</a> Stem Cells Dev. 17 (4), 653-667 (2008).</li><li>Dellavalle, A., 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=Pericytes+resident+in+postnatal+skeletal+muscle+differentiate+into+muscle+fibres+and+generate+satellite+cells.">Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells.</a> Nat Commun. 2, 499 (2011).</li><li>Mitchell, K. J., 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=Identification+and+characterization+of+a+non-satellite+cell+muscle+resident+progenitor+during+postnatal+development.">Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development.</a> Nat Cell Biol. 12 (3), 257-266 (2010).</li><li>Pannerec, A., Formicola, L., Besson, V., Marazzi, G., Sassoon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+skeletal+muscle+resident+progenitors+and+their+cell+fate+potentials.">Defining skeletal muscle resident progenitors and their cell fate potentials.</a> Development. 140 (14), 2879-2891 (2013).</li><li>Dellavalle, A., 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=Pericytes+of+human+skeletal+muscle+are+myogenic+precursors+distinct+from+satellite+cells.">Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.</a> Nat Cell Biol. 9 (3), 255-267 (2007).</li><li>Kostallari, 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=Pericytes+in+the+myovascular+niche+promote+post-natal+myofiber+growth+and+satellite+cell+quiescence.">Pericytes in the myovascular niche promote post-natal myofiber growth and satellite cell quiescence.</a> Development. 142 (7), 1242-1253 (2015).</li><li>Birbrair, A., 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=Role+of+pericytes+in+skeletal+muscle+regeneration+and+fat+accumulation.">Role of pericytes in skeletal muscle regeneration and fat accumulation.</a> Stem Cells Dev. 22 (16), 2298-2314 (2013).</li><li>Yao, Y., Norris, E. H., Mason, C. E., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+regulates+PDGFRbeta(+)+cell+stemness+and+muscle+development.">Laminin regulates PDGFRbeta(+) cell stemness and muscle development.</a> Nat Commun. 7, 11415 (2016).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Dore-Duffy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+pluripotent+cells+of+the+blood+brain+barrier.">Pericytes: pluripotent cells of the blood brain barrier.</a> Curr Pharm Des. 14 (16), 1581-1593 (2008).</li><li>Dore-Duffy, P., Katychev, A., Wang, X., Van Buren, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+microvascular+pericytes+exhibit+multipotential+stem+cell+activity.">CNS microvascular pericytes exhibit multipotential stem cell activity.</a> J Cereb Blood Flow Metab. 26 (5), 613-624 (2006).</li><li>Birbrair, A., 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=Skeletal+muscle+pericyte+subtypes+differ+in+their+differentiation+potential.">Skeletal muscle pericyte subtypes differ in their differentiation potential.</a> Stem Cell Res. 10 (1), 67-84 (2013).</li><li>Gautam, J., Nirwane, A., Yao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+differentially+regulates+the+stemness+of+type+I+and+type+II+pericytes.">Laminin differentially regulates the stemness of type I and type II pericytes.</a> Stem Cell Research &amp; Therapy. 8 (1), 28 (2017).</li><li>Birbrair, A., 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=Pericytes:+multitasking+cells+in+the+regeneration+of+injured,+diseased,+and+aged+skeletal+muscle.">Pericytes: multitasking cells in the regeneration of injured, diseased, and aged skeletal muscle.</a> Front Aging Neurosci. 6, 245 (2014).</li></ol>

모든 저자는 공개 할 이해 관계가 없습니다.

각질 세포는 모세 혈관 21 , 26 의 반 관절 표면에 위치한 다 능성 혈관 주위 세포 22 , 23 입니다. 골격근에서, 혈관 주위 세포는 지방 형성 및 / 또는 근원 형성 경로 19 , 20 , 24를 따라 분화 할 수 있습니다. 최근의 연구에 따르면 마커 발현이 ...

근이영양증은 지금까지 효과적인 치료법이없는 근육 퇴행성 장애입니다. 조직 재생을 촉진시키는 치료제 개발은 언제나 큰 관심사였습니다. 조직 재생 및 손상 후의 수리는 상주하는 줄기 세포 / 전구 세포에 달려 있습니다 1 . 위성 세포는 근육 재생 2 , 3 , 4 , 5 , 6 , 7에 기여하는 근원 전구 세포입니다. 그러나 그들의 임상 적 사용은 주사 후 제한된 이동 및 낮은 생존율뿐만 아니라 시험관 내 증폭 8 , 9 , 10 , 11 후에 감소 된 분화능에 의해 방해 받는다. satelli 외에도te 세포에서 골격근은 또한 혈소판 유래 성장 인자 수용체 - 베타 (PDGFRβ) - 양성 간질 세포와 같은 근육 형성 잠재력이 12 , 13 , 14 , 15 , 16 인 많은 다른 세포 집단을 포함한다. 근육에서 파생 된 PDGFRβ + 세포가 근원 세포로 분화되어 근육 병리학 및 기능을 향상시킬 수 있다는 증거가있다 14 , 17 , 18 , 19 , 20 . PDGFRβ는 우세하게 혈관 주위 세포 인 다 혈관 주위 세포 21 을 나타냅니다. 22 , 23 . PDGFRβ 외에도 Neuron-Glial 2 (NG2) 및 CD146을 비롯한 많은 다른 마커가 i혈관 주위 세포를 확인하십시오 21 . 그러나 이들 마커 중 어느 것도 둘 다 pericyte-specific 21 이 아님을주의해야합니다. 최근 연구에 의하면 유형 I과 유형 II라고 불리는 근육 주변 세포의 두 가지 유형이 밝혀 졌는데, 이는 서로 다른 분자 표지를 나타내며 별개의 기능을 수행합니다 19 , 24 , 25 . 생화학 적으로, 유형 I 주변 세포는 NG2 + Nestin - 이고, 유형 II 주변 세포는 NG2 + Nestin + 19 , 24 이다. 기능적으로 유형 I의 세포주는 지방 축적 및 / 또는 섬유화에 기여하는 지방 형성 분화를 겪을 수 있지만 유형 II의 혈관 주위 세포는 근원적 인 경로를 따라 분화되어 근육 재생에 기여할 수있다 19 , 24 , 25 . 이러한 결과는 다음을 입증합니다 : (1) 유형 I 혈소판은 b지방 퇴행성 장애 / 섬유증의 치료를 목표로하는, 및 (2) II 형 혈관 주위 세포는 근이영양증에 대한 치료 가능성이 크다. 이러한 개체군에 대한 추가 조사와 특성 규명을 위해서는 높은 수준의 순도에서 I 형 및 II 형 혈관 주위 세포의 분리를 가능하게하는 격리 프로토콜이 필요합니다. 현재, pericyte subpopulations의 고립은 NG2 - DsRed 및 Nestin - GFP 이중 transgenic 마우스 19 , 24 에 의존합니다. NG2-DsRed 생쥐의 유용성과 대부분의 NG2 항체의 품질은이 방법의 광범위한 사용을 제한합니다. 모든 NG2 + 혈관 주위 세포가 골격근 19 , 20 , 24 에서 PDGFRβ도 발현한다는 것을 감안할 때 우리는 혈관 주위 세포와 그 모집단의 분리를 위해 NG2가 PDGFRβ로 대체 될 수 있다고 가정한다. 이 작업은 FACS 기반 프로토콜을 설명합니다.PDGFRβ 염색 및 Nestin-GFP 신호를 사용합니다. 이 방법은 (1) NG2-DsRed 배경을 필요로하지 않으며 (2) 잘 특성화 된 상업적으로 이용 가능한 PDGFRβ 항체를 사용하기 때문에 조사자에게는 덜 까다로운 방법입니다. 또한 I 형과 II 형 혈관 주위 세포를 고순도로 동시에 분리 할 수있어 이러한 혈관 주위 세포 집단의 생물학적 특성과 치료 가능성을 연구 할 수 있습니다. 정화 후, 이들 세포는 배양 물에서 배양 될 수 있고, 그 형태는 시각화 될 수있다. 이 연구는 또한 이중 형질 전환 마우스에서 정제 된 것과 같은이 방법을 사용하여 분리 된 I 형 및 II 형 세포 주변 세포가 각각 지방 형성 및 근육 형성이라는 것을 보여줍니다.

<table><tbody><tr><td>Cell Sorter</td><td>Sony</td><td>SH800</td><td></td></tr><tr><td>Automatic Setup Beads</td><td>Sony</td><td>LE-B3001</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995</td><td></td></tr><tr><td>Avertin&amp;nbsp;</td><td>Sigma</td><td>T48402</td><td></td></tr><tr><td>Pericyte Growth Medium</td><td>ScienCell</td><td>1201</td><td></td></tr><tr><td>MSC Basal Medium (Mouse)</td><td>Stemcell Technologies</td><td>5501</td><td></td></tr><tr><td>Adipogenic Stimulatory Supplement (Mouse)</td><td>Stemcell Technologies</td><td>5503</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000</td><td></td></tr><tr><td>Horse Serum</td><td>Sigma</td><td>H1270</td><td></td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>0.25% Trypsin/EDTA&amp;nbsp;</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>PDL</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>PDGFR&amp;beta;-PE Antibody</td><td>eBioscience</td><td>12-1402</td><td></td></tr><tr><td>Perilipin Antibody</td><td>Sigma</td><td>P1998</td><td></td></tr><tr><td>S-Myosin Antibody</td><td>DSHB</td><td>MF-20</td><td></td></tr><tr><td>Alexa 555-anti-rabbit antibody&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>A-31572</td><td></td></tr><tr><td>Alexa 555-anti-mouse antibody</td><td>ThermoFisher Scientific</td><td>A-31570</td><td></td></tr><tr><td>Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2058</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Fisher Scientific</td><td>A661</td><td></td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P184</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190</td><td></td></tr><tr><td>18 G Needles</td><td>BD</td><td>305196</td><td></td></tr><tr><td>10 mL Serological Pipette</td><td>BD</td><td>357551</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111</td><td></td></tr></tbody></table>

isolation of type i and type ii pericytes from mouse skeletal muscles

각질 세포는 여러 기관 또는 심지어 같은 조직 내에서 이질성을 나타내는 혈관 주변의 다 분화능 세포입니다. 골격근에는 여러 분자 마커를 표현하고 뚜렷한 분화능을 갖는 적어도 2 개의 일주 체형 하위 집단 (유형 I 및 유형 II라고 함)이 있습니다. NG2-DsRed 및 Nestin-GFP 이중 - 형질 전환 마우스를 사용하여, 유형 I (NG2-DsRed + Nestin-GFP-) 및 유형 II (NG2-DsRed + Nestin-GFP + ) 혈관 주위 세포가 성공적으로 분리되었다. 그러나 이러한 double-transgenic mice의 유용성은이 정제 방법의 광범위한 사용을 방해합니다. 이 연구는 골격근으로부터 I 형 및 II 형 세포를 쉽고 동시에 분리 할 수있는 대체 프로토콜을 설명합니다. 이 프로토콜은 형광 활성화 세포 정렬 (FACS) 기술을 이용하고 Nestin-GFP 신호와 함께 NG2보다는 PDGFRβ를 표적으로 삼습니다. 격리 후 I 및 ty를 입력하십시오.pe II 혈관 주위 세포는 별개의 형태를 나타낸다. 또한 이중 형질 전환 마우스에서 분리 된 것과 같은이 새로운 방법으로 분리 된 1 형 및 2 형 혈관 주위 세포는 각각 지방 형성 및 근육 형성이다. 이 결과는이 프로토콜이 골격근과 아마도 다른 조직으로부터 일주 전 세포 모집단을 분리하는데 사용될 수 있음을 시사한다.

레이저 강도 및 채널 보정을 포함한 FACS 매개 변수는 염색되지 않은 컨트롤 및 단색 컨트롤의 결과를 기반으로 보정됩니다. PDGFRβ-PE-FMO 대조군은 PDGFRβ-PE + 집단 ( 그림 1A )에 대한 게이팅을 설정하는 데 사용됩니다. PDGFRβ-PE 세포 중 Nestin-GFP + 와 Nestin-GFP - 세포를 나타내는 두 집단이 명확하게 분리되어있다 ( ?...

Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

이 연구는 골격근에서 I 형 및 II 형 혈관 주위 세포를 쉽고 동시에 격리 할 수있는 FACS 기반 프로토콜에 대해 설명합니다.

마우스 골격근에서 I 형과 II 형 Pericytes 분리

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

isolation-of-type-i-and-type-ii-pericytes-from-mouse-skeletal-muscles

Research

JoVE Journal

Developmental Biology

9.1K 조회수.  University of Minnesota.  이 연구는 골격근에서 I 형 및 II 형 혈관 주위 세포를 쉽고 동시에 격리 할 수있는 FACS 기반 프로토콜에 대해 설명합니다. 

비디오: Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

인간의 골격근에서 기본 근육 조직 세포와 섬유 아 세포의 분리 및 정량 면역 세포 화학 특성

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

연구

발생학

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

마우스 골격 근 이식에서 기본 근구의 격리 및 분화

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

기본 Murine 골격 근육 Microvascular 내 피 세포의 고립

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

신경과학

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 ...

생물학

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

근육 위성 세포의 분리, 문화, 및 이식

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

신생아 쥐의 폐 동맥 평활근 세포의 분리

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

뮤린 심장 pericytes의 격리 및 정화

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

의학

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

초기 산후 발달에 있는 에머리 드레이푸스 근 이영양증의 Murine 모형에서 단 근섬유 격리 및 문화

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

마우스에서 대뇌 골후혈구를 분리하는 고출력 방법

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation assay.

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2&#180;-deoxyuridine incorporation assay.

개별 골격근에서 정지 줄기 세포 집단의 분리

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...