이 연구는 미국 심장 협회 부여 0950118G.to MC - W에 의해 부분적으로 지원되었다.

1. 안티 PECAM - 1 항체 - 복합 자석 구슬을 준비 (Dynabeads) <ol><li> BSA가 해소 때까지 소용돌이를 사용하여 50 ML PBS에서 50 MG BSA를 혼합하여 PBS에 0.​​1 % BSA를 준비합니다. 0.22 μm의 주사기 필터를 통해 필터링하여 소독. 4 스토어 ° C. </li><li> TC 후드에서 1.5 ML Eppendorf 튜브에 잘 resuspended 양을 안티 쥐 IgG의 Dynabeads 200 μl를 전송하고 구슬을 씻으 : 마운트 튜브를 MPC와 퇴적물에 비즈를 허용하도록 약 1 분 앉아 보자. 뜨는 기음, MPC에서 튜브를 제거하고 멸균 0.1 % BSA / PBS 1 ML에있는 구슬을 resuspend. 구슬을 resuspend하기 위해 아래 피펫합니다. </li><li> 네 세척의 총 세 번 씻어 반복합니다. </li><li> MPC에서 튜브를 제거하고 0.1 % BSA / PBS 500 μl의 구슬을 resuspend. </li><li> 튜브 10 μl 쥐 방지 마우스 PECAM - 1 (CD - 31) 항체를 추가합니다. </li><li> 상온에서 추운 방 또는 2 시간에서 4 ° C에서 하룻밤 텀블. </li><li> 같은 단계 1.2 네 번에서 설명한 멸균 0.1 % BSA / PBS로 비즈를 씻으십시오. </li><li> MPC에서 튜브를 제거하고 200 μl 0.1 % BSA / PBS에있는 구슬을 resuspend. 4 스토어 방지 PECAM - 1 항체 - 복합 Dynabeads 최대 일개월하는 C. </li></ol> 2. 신생아 생쥐에서 분리 폐 내피 세포 마우스 전에 조직 정화 프로토콜과 함께 진행하는 프로 시저의 IACUC위원회의 승인이 필요합니다. <ol><li> 다음 사항을 준비 : <ul><li> DMEM 1 MG / ML C / D 솔루션 15 ML. 37 필터 따뜻한 0.22 μm의 주사기와 필터 ° C. </li><li> 50 ML 원뿔 튜브 15 ML DMEM, 얼음에 저장과 </li><li> DMEM에 최고 20 % FBS 및 1X 페니실린 / 스트렙토 마이신을 포함한 절연 미디어 (IM) </li><li> 젤라틴 2%. 4 100 ML DD 물, 압력솥 15 분 저장 ° C.와 소 피부 2g는 ​​젤라틴 믹스 37 따뜻한 ° C 코팅 TC의 flasks 전에 녹일 수 있습니다. </li></ul></li><li> 케타민을 (100 MG / kg)과 xylazine (10 MG / kg)의 IP 주사로 3 6~8일 오래된 새끼를 마취. 다음과 같이 마취의 효과를 확인하고 동물 하나씩 해부하다 : 보드에 핀 새끼는 멸균 해부 가위로 가슴 영역에서 피부를 70 % 에탄올로 피부를 흡수하고 제거합니다. 폐가 노출 엑사이스 갈빗대. </li><li> 무균 엑사이스 개별 폐 엽 (叶), 조심하는 것은 기관지와 폐 주변에 보이는 결합 조직을 해부하다하지. 얼음처럼 차가운 DMEM의 모든 폐를 결합합니다. 새끼를 안락사. </li><li> TC 후드에서 근무 DMEM, 100mm TC 요리의 장소에서 폐 엽 (叶)을 제거하고 여분의 DMEM을 대기음. </li><li> 멸균 가위를 사용하여 ~ 100 번 절단하여 가늘게 조직을 말하다. 효소 소화 15 ML 따뜻한 C / D 솔루션에 전송합니다. 회전에서 37 ° C에서 45 분 알을 품다. </li><li> TC 후드에서 단일 세포 현탁액에 14g​​ 정맥 붙어 씹다 대단히 짧은 시간, 최소한 12 시간을 20 ML의 주사기로 들어가서 소화 조직 현탁액을 대기음. </li><li> 70 μm의 셀 스트레이너를 통해 조직 현탁액을 통과하고 소화를 중지 15 ML 메신저로 스트레이너 씻으십시오. </li><li> 5 분 400x g에서 원심 분리하여 펠렛의 세포 현탁액. </li><li> 3 ML 0.1 % BSA / PBS에 대기음 뜨는 및 resuspend 펠릿. </li><li> 5 ML 둥근 바닥 폴리스티렌 튜브에 세포 현탁액을 전송하고 22.5 μl 방지 PECAM - 1 항체 - 복합 Dynabeads를 추가합니다. 12분에 대한 실온에서 텀블. </li><li> 코트 2 ML 2퍼센트 젤라틴과 젤라틴 대기음 초과와 T75 플라스크. 이전 도금 세포 ~ 30 분 정도에 대한 TC 후드 내부 건조 젤라틴 수 있습니다. </li><li> 비드 배양이 완료되면 (단계 2.10) 세 Eppendorf 튜브에 동일 세포 현탁액을 분할하여 MPC에 탑재합니다. </li><li> 구슬은 MPC를 (~ 1 분)를 사용하여 sedimented있을 때, 뜨는 수집 MPC에서 튜브를 제거와 구슬을 씻어 ~ 1 ML 0.1 % BSA / PBS 다음 MPC를 탑재. </li><li> 네번 (총 5 세척)을 씻어 반복 </li><li> EnGS - MV 생활 요인 Kit 및 관 당 1X 페니실린 / 스트렙토 마이신 (VL)와 VascuLife 1 ML에 Resuspend 구슬이 잘 혼합. </li><li> 미리 코팅 T75 플라스크에 플레이트와 VL 10 ML 각 플라스크 총 볼륨을 가지고. </li><li> 미디어에게 다음날을 변경, 성장의 한 하루를 허용하고 다른 모든 일 미디어의 절반을 변경합니다. 세포 (보통 문화의 3~4일 후)&gt; 70-80% 합류 이상의 경우, 그들은 반 ICAM - 2 Dynabeads와 함께 정렬할 수 있습니다. </li></ol> 3. 준비 방지 ICAM - 2 항체 - 복합 Dynabeads <ol><li> 안티 ICAM - 2 항체 - 복합 Dynabeads가 추가 합류 때까지 반 PECAM - 1 Dynabeads 항체 - 복합과 교양과 선택되어 세포를 정화하는 데 사용됩니다. 이 절차는 그 반대 마우스 ICAM - 2 (CD - 102)가 항체가 백신 PECAM - 1 항체 대신 사용 제외하고, 안티 PECAM - 1 항체 - 복합 Dynabeads (1 위)에 대한 설명으로 수행됩니다. </li><li> 안티 ICAM - 2 항체 - 복합 Dynabeads는 ° C 최대 4 저장할 수 있습니다일개월 수 있습니다. </li></ol> 4. 안티 ICAM - 2 Dynabeads와 마우스 폐 내피 세포 정렬 <ol><li> 2퍼센트 젤라틴과 코트 T75 조직 문화 플라스크. </li><li> 세포와 합류 T75 술병에서 미디어를 대기음 8 ML PBS로 세척. </li><li> 대기음 PBS, 2 ML 0.05 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)을 추가하고 (5 분) 세포가 분리하자. 분리 원조로 가볍게 flasks을 누릅니다. </li><li> 세포가 분리되면 남아있는 자기편 세포를 분리하는 트립신과 다쳤고, 세포를 억제 2 ML IM를 추가합니다. 15 ML 튜브에 세포 현탁액을 전송하고 400x G.에서 5 분 스핀 다운 </li><li> 2 ML 0.1 % BSA / PBS에있는 미디어 및 resuspend 세포 펠렛을 대기음. </li><li> 5 ML의 폴리스티렌 왕복 아래 튜브로 환승 10 μl 방지 ICAM - 2 Dynabeads를 추가합니다. RT 12 분 텀블. </li><li> MPC 두 1.5 ML Eppendorf 튜브 및 마운트에 세포 현탁액을 분할. </li><li> 구슬 한 분만 준수 후에 뜨는을 대기음. MPC에서 튜브를 제거하고 1 ML 0.1 % BSA / PBS에 각 튜브를 resuspend. MPC에 리마운트. </li><li> 네 번 (5 씻어 총) 씻어 반복합니다. </li><li> 1 ML VL 각 튜브를 Resuspend 및 셀 개수를 수행합니다. </li><li> T25 플라스크 및 / 또는 T75 플라스크 당 750K - 1000000 세포 당 250 350K 전지에서 플레이트 세포. </li><li> T25 flasks 5 ML의 VL, 그리고 T75 flasks 10 ML에 볼륨을 가져와. </li><li> 매일 미디어를 변경합니다. 문화가 보통 2~3일에 약 80 % confluency에 도달하면 세포는 실험을 위해 사용될 수 있습니다. </li></ol> 5. 대표 결과 일반적으로 세포의 초기 준비 6-7일 후, 우리는 1.2에 대해 얻을 수 있습니다 - 셋 6~8일 오래된 새끼에서 1.5 X 10 6 폐 내피 세포는. 세포는 빛을 현미경 아래에 전형적인 &quot;조약돌&quot;형태를 표시하고 내피 세포 (그림 1)에 대한 특징입니다 VE - cadherin (CD144) 세포 세포 분기점에서 얼룩을 보여줍니다. MLECs 표현 VE - cadherin과 VEGFR2의 대부분은 외과 분석 (그림 2)에서 보여주었다. 보통, 우리는 초기 MLEC 격리 이후 두 주 이내에 실험에 사용합니다. <img src="/files/ftp_upload/2316/2316fig1.jpg" alt="그림 1" /> 그림 1. 합류 MLEC의 monolayer의 미세 분석. (A) 라이트 현미경 이미지는 내피 세포에 대한 전형적인 교양 세포의 조약돌 형태를 보여줍니다. 많은 세포의 perinuclear 지역에 위치하고 균일한 원형 구조는 MLEC 절연에 사용되는 자성 비즈입니다. (B) 내피 특정 VE - cadherin은 같은 공촛점 현미경을 사용하여 표시된 셀 전지 분기점에서화된입니다. 바 20μm의 대표입니다. <img src="/files/ftp_upload/2316/2316fig2.jpg" alt="그림 2" /> 그림 2 내피 특정 마커에 대한 특정 항체로 분류 교양 MLECs의 외과 분석 :. phycoerythrin - 복합 이차 항체 다음 VE - cadherin (CD144) 또는 VEGFR2로 기소, 절연 MLECs의 내피 신원을 확인합니다. 레드 라인 isotype 특정 컨트롤을 나타냅니다, 녹색 라인은 나타냅니다 방지 VE - cadherin 또는 방지 VEGFR2 - 특정 - IgG.

<ol>
	<li>Maruyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+endothelial+cell+in+tissue+culture.">The human endothelial cell in tissue culture.</a> Z Zellforsch Mikrosk. Anat. 60, 69-79 (1963).</li><li>Lim, Y. C., Luscinskas, 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=Isolation+and+culture+of+murine+heart+and+lung+endothelial+cells+for+in+vitro+model+systems.">Isolation and culture of murine heart and lung endothelial cells for in vitro model systems.</a> Methods Mol Biol. 341, 141-154 (2006).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circ Res. 100, 158-173 (2007).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., Delisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 296, L1096-L1103 (2009).</li><li>Miebach, S., Grau, S., Hummel, V., Rieckmann, P., Tonn, J. C., Goldbrunner, R. H. <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+culture+of+microvascular+endothelial+cells+from+gliomas+of+different+WHO+grades.">Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades.</a> J. Neurooncol. 76, 39-48 (2006).</li><li>Demeule, M., Labelle, M., R&#233;gina, A., Berthelet, F., B&#233;liveau, R. <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+endothelial+cells+from+brain,+lung,+and+kidney:+expression+of+the+multidrug+resistance+P-glycoprotein+isoforms.">Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms.</a> Biochem. Biophys. Res. Commun. 281, 827-834 (2001).</li><li>Kajimoto, K., Hossen, N., Hida, K., Ohga, N., Akita, H., Hyodo, M., Hida, Y., Harashima, H. <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+culture+of+microvascular+endothelial+cells+from+murine+inguinal+and+epididymal+adipose+tissues.">Isolation and culture of microvascular endothelial cells from murine inguinal and epididymal adipose tissues.</a> J. Immunol. Methods. 357, 43-50 (2010).</li><li>Breviario, F., Caveda, L., Corada, M., Martin-Padura, I., Navarro, P., Golay, J., Introna, M., Gulino, D., Lampugnani, M. G., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+human+vascular+endothelial+cadherin+(7B4/cadherin-5),+an+endothelium-specific+cadherin.">Functional properties of human vascular endothelial cadherin (7B4/cadherin-5), an endothelium-specific cadherin.</a> Arterioscler Thromb Vasc Biol. 15, 1229-1239 (1995).</li><li>Zachary, I., Gliki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+transduction+mechanisms+mediating+biological+actions+of+the+vascular+endothelial+growth+factor+family.">Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family.</a> Cardiovasc Res. 49, 568-581 (2001).</li><li>Diglio, C. A., Grammas, P., Giacomelli, F., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+heart-derived+endothelial+and+smooth+muscle+cell+cultures:+isolation,+cloning+and+characterization.">Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.</a> Tissue Cell. 20, 477-492 (1988).</li><li>Petzelbauer, P., Bender, J. R., Wilson, J., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J Immunol. 151, 5062-5072 (1993).</li><li>Chrzanowska-Wodnicka, M., Kraus, A. E., Gale, D., White, G. C., Vansluys, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+angiogenesis,+endothelial+migration,+proliferation,+and+MAPK+signaling+in+Rap1b-deficient+mice.">Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice.</a> Blood. 111, 2647-2656 (2008).</li></ol>

동물 실험 프로토콜 AUA # 00001206 이하 위스콘신 IACUC위원회의 메디컬 칼리지에 의해 명시된 지침과 규정에 따라 수행되었다.

Microvascular 항법은 혈관 생물학의 여러 분야에서 유용한 모델로 입증하고 널리 공부 HUVECs 세 이상 (예 : angiogenesis) 공부에 더 생리학 관련성이 높은 것으로하고 있습니다. 이전, 그것은 microvascular 내장 컴퓨터가 신장, 심장, 피부, 망막, 뇌, gliomas, 지방 조직뿐만 아니라 폐 2, 4-7, 10, 11에서 구할 수있는 것으로보고되었습니다. 그러나, microvascular 내장 컴퓨터의 격리에 대한 일관성 있고 신뢰할 수있는 방법은 여전히​​ 필요합니다. 여기에 제시 절차는 이전에 게시된 프로토콜 2의 수정이며 그것이 새끼 대신 성인 동물을 사용하는 대부분의 출판 절차와는 다릅니다. 어린 동물에서 세포가 높은 확산 가능성을 가지고 자신의 조직에서 파생된 문화가 세포의 높은 숫자를 얻을하는 경향으로 이것은 절연 성공을 위해 매우 중요합니다. 일주일 오래된 동물 최적으로 보이지만, 어린 생쥐 (하루 지난 4)도 사용할 수 있습니다. 우리는 하나 펍에서 MLECs를 분리에 성공으로도, 새끼의 높거나 낮은 숫자는 필요한 경우 사용할 수 있습니다. 그러나, 격리 프로세스를 확장 또는 아래 반면, 세포 도금 밀도는 또한 세포 증식에​​ 중요로 변경되어 있어야합니다. 안티 PECAM - 1 최초로 복합 자기 구슬을 사용하여 MLECs 및 방지 ICAM - 2 항체보다 2 단계 정화 과정은 앞에서 설명한 단일 단계 정화 절차를보다 순수한 EC 문화를 얻는에 훨씬 효율적입니다. 이 프로토콜은 매우 힘드는 매뉴얼 기술, 기울기 원심 분리 및 섬유아 세포, 혈액 세포 및 평활근 세포로 폐기 오염 전지 정렬 덜 효율적인 외과를 사용할 필요가 없습니다. 그 결과 MLEC 인구는 angiogenic 반응, 혈관 투과성 및 백혈구 윤회의 체외 분석, 치료뿐만 아니라 신호 경로의 생화 학적 분석을 상처를 사용할 수 있습니다. 필요한 경우, MLECs 그러한 횡단 내피 저항 (터) 측정 immunofluorescence 또는 전극 어레이에 대한 fibronectin이나 젤라틴 코팅 coverslips 등 다양한 표면에 도금 수 있습니다. 얻은 결과는 직접 녹아웃 및 유전자 변형 마우스 라인의 증가의 맥락에서 특히 중요합니다 생체내에서 관찰 phenotypes 비교할 수 있습니다. 생체내에서 관찰된 결함을 기본 세포 구조의 체외 분석에이 방법의 성공적인 구현은 이미 12를 발표했습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15032-13</td>
<td>&nbsp;</td>
<tr>
<td>forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11223-20</td>
<td>&nbsp;</td>
<tr>
<td>14G cannula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482516N</td>
<td>&nbsp;</td>
<tr>
<td>20 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309661</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>A7030-100G</td>
<td>&nbsp;</td>
<tr>
<td>anti-rat IgG Dynabeads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.35</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Particle Concentrator (MPC)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse PECAM-1 antibody (CD-31)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553369</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse ICAM-2 antibody (CD-102) </td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553326</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase/Dispase (C/D)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11097113001</td>
<td>100mg/ml stock solution can be prepared and stored at -20&deg;C</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Skin Gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#G9391-100G</td>
<td>&nbsp;</td>
<tr>
<td>Vasculife EnGS-Mv complete kit (VL)</td>
<td>&nbsp;</td>
<td>Lifeline Cell Technology</td>
<td>LL0004</td>
<td>&nbsp;</td>
<tr>
<td>0.05% Trypsin/EDTA </td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer 70 &mu;m nylon</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>tissue culture flask 75 cm2</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>90076</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of pulmonary endothelial cells from neonatal mice

내피 세포는 혈관 생물학의 여러 분야에서 유용한 연구 모델을 제공합니다. 처음으로 고립 1 일 이후, 인간 제대 정맥 내피 세포 (HUVECs) 가장 널리 연구 내피 세포 수 있습니다 따라서, 구하는 문화를 쉽고 편리하게하는 표시하고 있습니다. 그러나, angiogenesis, 투자율이나 많은 사람과 같은 프로세스에 초점을 맞춘 연구, microvascular 내피 세포 (내장 컴퓨터 시스템)은 2 연구 훨씬 더 생리학 관련성이 높은 모델입니다. 또한, 녹아웃 마우스에서 분리된 내장 컴퓨터 단백질 기능의 예 생체내 분석을위한 유용한 도구를 제공합니다. 몇 가지 접근 방법은 격리와 문화 microvascular 다른 원산지의 항법 것은 날짜를 3-7로보고 있지만, 순수 항법의 일관성 고립과 문화는 여전히 많은 실험실에서 중요한 기술적 문제되었습니다. 여기서 우리는 분리하고 culturing 마우스 폐 내피 세포 (MLECs)의 안정적이고 비교적 간단한 방법에 대한 단계별 절차를 제공합니다. 이 방법에서는 폐 조직은 6에서 얻은 - 오래된 새끼가 처음 collagenase / dispase (C / D) 솔루션과 함께 소화, 조각으로 절단 및 단일 세포 현탁액으로 기계적 분산됩니다 8 일 수 있습니다. MLECS은 자기 입자 농축기 (MPC)를 사용 Dynabeads에 복합 백신 PECAM - 1 항체와 긍정적인 선택을 사용하여 세포 현탁액에서 정화 수 있습니다. 그들이 합류되기 전까지는 이러한 정화 세포 (TC) 요리 젤라틴 코팅 조직 문화에 양식 있습니다. 그 시점에서, 세포는 더 이상 반 ICAM - 2 항체에 결합 Dynabeads를 사용하여 정화하고 있습니다. 이 프로토콜과 함께 얻은 MLECs는 위상 대비 가벼운 현미경에 의해 같은 시각 코블스톤 표현형을 전시, 그들의 내피 표현형는 안티 - VE - cadherin 8 방지 VEGFR2 9 항체 및 VE - cadherin의 immunofluorescent 얼룩과 외과 분석을 사용하여 확인되었습니다. 우리 손에,이 두 단계 절연 절차는 지속적으로 안정 추가로 교양 수 MLECs의 순수한 인구를 산출. 이 방법은 연구자가 녹아웃 직접 격리 MLECs에서 수행 체외 실험에서 결과와 생체내 연구에 연관시키는 유전자 변형 생쥐의 증가를 활용하기 때문에 생체내에서 관찰 혈관 phenotypes의 분자 메커니즘을 공개하는 데 도움이있게됩니다.

Watch this Scientific Journal Video about Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice at JoVE.com

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

여기, 우리는 murine 폐동맥 내피 세포의 격리와 문화 프로토콜을 설명합니다. 이 방법은 정비사 및 효소 폐 조직 분리뿐만 아니라 안티 - PECAM - 1과 대부분 microvascular 원산지 순수한 내피 세포 인구를 생산 자성 비즈에 복합 백신 ICAM - 2 항체를 사용하여 2 단계 정화 과정으로 구성됩니다.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

isolation-culture-pulmonary-endothelial-cells-from-neonatal

Research

JoVE Journal

Biology

47.0K 조회수.  BloodCenter of Wisconsin. 여기, 우리는 murine 폐동맥 내피 세포의 격리와 문화 프로토콜을 설명합니다. 이 방법은 정비사 및 효소 폐 조직 분리뿐만 아니라 안티 - PECAM - 1과 대부분 microvascular 원산지 순수한 내피 세포 인구를 생산 자성 비즈에 복합 백신 ICAM - 2 항체를 사용하여 2 단계 정화 과정으로 구성됩니다.

비디오: Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

판막병 내피 세포의 분리

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

연구

생물학

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

분리 및 문화 신생아 마우스 심근

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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 of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

쥐과 밸브 내피 세포의 분리

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

분리 및 마우스 대동맥 내피 세포의 차 문화

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

사람 말초 혈액에서 생성 및 문화 혈액의 파생물 내피 세포

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

마우스 관상 내피 세포의 분리

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

소형 돼지에서 1 차 대동맥 내피 세포의 격리 및 배양

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...