JSO는 NIH 루스 L. 키르 NRSA (F32 DK091040)에 의해 지원됩니다, AK는 미국 당뇨병 협회 (7-10-MI-05)에서 박사후 원정대에 의해 지원되며, AHH는 미국 심장 협회 설립 탐정 수상에 의해 지원됩니다 (12EIA8270000). 유동 세포 계측법 실험은 VMC 유동 세포 계측법 공유 리소스에서 수행 하였다. VMC 유동 세포 계측법 공유 리소스는 밴더빌트 소화기 질환 연구 센터 (DK058404)에 의해 지원됩니다.

1. 시약 및 소모품 앞서 실험 프로토콜을 시작하려면 다음 시약을 준비 : <ol><li> 70 % 에탄올 </li><li> 1X PBS </li><li> 1X DPBS은 (칼슘과 마그네슘 제외) 0.5 % BSA가 첨가 </li><li> FACS 버퍼 : 1X DPBS (칼슘과 마그네슘 제외), 2 mM의 EDTA (에틸렌 다이아 민 테트라 초산), 1 % FCS </li><li> ACK 버퍼 : 150 MM의 NH 4 망할 CIA, 10 MM KHCO 3, 물에 0.1 밀리미터 나 2 EDTA (에틸렌 다이아 민 테트라 초산) </li></ol> 2. 지방 조직의 수확 및 준비 <ol><li> 각 기관에 특정한 IACUC 승인 된 절차에 따라 쥐를 안락사. </li><li> 철저하게 70 % 에탄올로 모피 젖은. </li><li> 검상 돌기 프로세스의 수준 (흉골의 하부)에서 절개를하고 모든 주요 혈관을 절단되지 않도록주의, 심장을 노출 흉강을 엽니 다. </li></ol> 참고 :이 시점에서만큼 진동판이 그대로 유지하는 것도 도움이 그 것이다가능한 혈액을 허용하고 흉강에서 흘러 관류하는 흉곽의 오른쪽의 노치를 잘라합니다. <ol start="4"><li> 혈액을 허용하고 순환 시스템을 탈출하기 위해 관류하는 우심방 클립. </li></ol> 참고 : AT 비만 쥐, 과도한 심낭을 처리 할 때 심장에 대한 액세스를 허용하기 위해 제거해야 할 수도 있습니다. <ol start="5"><li> 집게와 함께 마음을 잡고 조심스럽게 정점을 통해 좌심실에 바늘을 삽입합니다. 천천히 15 ML 멸균 PBS와 마우스를 붓는다. </li></ol> 참고 : 폐 작성하고 확장하기 시작하면 혈류의 속도를 줄입니다. <ol start="6"><li> 복강을 열고있는 성선 조직을 피하기 위해주의를 사용하여 perigonadal 지방 패드를 제거합니다. </li><li> 지방 패드를 배치하는 것은 0.5 % BSA가 첨가 2 ML 1X DPBS을 (마그네슘 또는 칼슘없이) 포함 얼음에 보트를 달아 정밀한 조각으로 AT를 말하다. </li></ol>참고 : 당 AT의 양을 제한 1.2 g에 배를 무게. AT의 양이 1.2 g을 초과하는 경우, 두 개의 무게 보트 사이에 균등하게 나눕니다. <ol start="8"><li> 얼음에 샘플 AT 유지하고 0.5 % BSA, 10 MM 염화칼슘 2, 4 MG / ML 콜라, 유형 II와 보충 1X DPBS로 구성된 샘플을 당 ML 콜라 다이제스트 솔루션을 준비합니다. </li></ol> 3. 콜라게나 소화 <ol><li> 균질를 따르고, 1 ML의 DPBS (0.5 % BSA) 3 ML 콜라 II 다이제스트 솔루션 보트의 무게를 세척하여 50 ML 원뿔 튜브로 전송할 수 있습니다. </li><li> 20 분 37 회전 흔드는 (200 RPM) ° C에서 균질 품다. </li><li> 얼음에 원뿔 튜브와 장소에 10 ML의 DPBS를 (0.5 % BSA)를 추가합니다. </li><li> 10 ㎖ 혈청 피펫을 사용하여 여러 번 균질 씹다, 새로운 50 ML 원뿔 관에 100 μm의 필터를 통해 세포 현탁액을 전달합니다. </li><li> 4 &amp; D에서 10 분 500 XG에서 세포 현탁액을 원심 분리기예를 들어, C. </li><li> 가만히 따르다 뜨는을 Resuspend SVF 세포 펠렛 3 ML ACK 버퍼에 적혈구의 오염을 용해합니다. </li><li> 4시 10 분 500 XG에 12 ML의 FACS 버퍼와 원심 분리기 세포 현탁액을 추가 ° C. </li><li> FACS 버퍼에서 가만히 따르다 뜨는을 Resuspend SVF 세포 펠렛. </li></ol> 참고 : 세포 펠렛은 50 ML 원뿔 튜브의 바닥을 커버하는 경우, 0.5 ML 외과 버퍼를 사용 인치 resuspend을하는 방법에 많은 FACS 버퍼에 대한 가이드로 세포 펠렛의 크기를 사용하고, 그렇지 않은 경우 0.25-0.5에 resuspend을 ML. <ol start="9"><li> 장소 얼음 샘플 및 혼합 40 μL FACS 버퍼 50 μL 판 블루 용액 (0.2 %), 10 μL 세포 현탁액으로 세포 계수에 대한 각 시료 1:10으로 희석 분주을 준비합니다. </li><li> 판 블루 배제에 따라 가능한 세포를 계산하고 5-10 X 10 6 세포 / ML의 최종 농도로 세포 현탁액을 희석. </li></ol> 4. 세포 표면 항원 염색 <oL&gt; <li> 0.5 μg/10 6 세포의 최종 농도 항 - 마우스 CD16/CD32 항체 (FC 블록)을 추가하고 10 분 동안 얼음에 품어. </li><li> 전송 샘플 (≥ 10 6 세포) 12 X 75mm의 폴리스티렌 원형 바닥 튜브. 현금 지급기와 T 세포를 분석하면 별도의 튜브를 준비하고, 필요한 보상 및 수정 형광 뺀 (FMO) 컨트롤을 수용하기 위해 튜브의 적절한 수를 준비하는 여분의 세포를 결합합니다. </li></ol> 참고 : 예를 들어, F4/80 및 CD11b, 다음과 같은 보상 및 FMO 컨트롤에 따라 현금 인출기의 비율을 정량화 할 때하는 준비를해야합니다 : - 흠 (세포) - DAPI 또는의 propidium 요오드화 물의 (PI) 단일 얼룩 (세포, 단계 5.1까지 생존 염료를 추가하지 마십시오) - F4/80 APC 얼룩 하나 (세포 나 보상 비즈) - CD11b FITC (얼룩 단일 세포 또는보상 비즈) - FMO 1 (셀) : 쥐 IgG2a κ 이소 타입 제어 APC + CD11b FITC + 생존 염료 (단계 5.1에 추가) - FMO 2 (셀) : F4/80 APC + 쥐 IgG2b κ 이소 타입 제어 FITC + 생존 염료 (단계 5.1에 추가) <ol start="3"><li> (시약 및 재료의 표 참조) 적절한 농도에서 형광 - 복합 일차 항체 및 / 또는 이소 타입의 컨트롤을 추가합니다. </li><li> 30 분 동안 4 ° C에서 빛과 부화에서 샘플을 보호합니다. </li><li> 4시 5 분 500 XG에 2 ㎖의 FACS 버퍼와 원심 분리기 세포 현탁액을 추가 ° C. </li><li> 2 ㎖ FACS 버퍼에서 가만히 따르다 뜨는을 Resuspend SVF 세포 펠렛. </li><li> ≥ 400 μL FACS 버퍼에 5 ° C에서의 분, 및을 Resuspend SVF 세포 펠렛을 위해 세포 현탁액 500 XG에 원심 분리기. </li><li> 35 μm의 셀 스트레이너 관 정상을 갖춘 12 X 75mm의 폴리스티렌 원형 바닥 튜브에 샘플을 전송합니다.</li><li> 4에 빛 및 저장 샘플로부터 보호 ° C를 FACS 분석까지. </li></ol> 참고 : 최적의 결과를 세포 immeadiately 분석해야하지만,이 프로토콜 FACS 분석을 성공적으로 4 ℃에서 1-2 시간 동안 FACS 버퍼에 저장된 라벨 세포에서 수행되었다 세포는 저장 시간이 증가하도록 수정해야 할 경우, 레이블이 세포는 FACS 분석을하기 전에 24 시간 동안 4 2 % 파라 포름 알데히드 ° C로 고정 할 수 있습니다. 항체 회사라는 세포가 1 주일로 저장할 수 있습니다하는 것이 좋습니다 있지만, 이것은이 절차의 컨텍스트 내에서 테스트되지 않았습니다. 5. FACS 분석 <ol><li> 이전 FACS 분석에 죽은 / 라이브 셀 차별을 허용하도록 샘플 및 적절한 컨트롤에 생존 염료를 추가합니다. </li></ol> 참고 : 많은 가능성 염료 상업적으로 사용할 수 있지만, DAPI 및 PI는 여기 / 방출 교수에 따라 추천형광 - 복합 항체의 레스가 사용되고있다. DAPI와의 propidium 요오드화물은 0.2 밀리그램 / ML의 최종 농도에서 각각의 샘플에 추가됩니다. <ol start="2"><li> 관심의 세포 인구 (들) 규모가되도록 측면 산란 (SSC) 앞으로 분산 형 (FSC)를 조정하는 실험 샘플과 같은 조직에서 고립 된 흠없는 대조군 샘플, 바람직 세포를 사용합니다. </li></ol> 참고 : SVF 세포 AT의 분석을 위해, 그것은 SSC는 리니어 스케일에 FSC 대 로그 스케일로 표시하는 것이 좋습니다. 종종 매우 크고 세분화되어 현금 인출기를 분석 할 때 SSC에 대한 로그 스케일의 사용은 특히 중요합니다. <ol start="3"><li> 분석되고 셀 (들)의 유형에 따라 초기 빛의 산란 문을 그리고 흠없는 세포까지 (약 10 2)를 중심으로 단일 매개 변수 히스토그램의 왼쪽에 위치하도록 광전 튜브 (PMT)는 게인 조정 해당 채널. &lt;/ 리&gt; </ol> 주 : 림프구와 대 식세포 (또는 다른 골수 세포) 때문에자가 형광의 차이에 개별적으로 분석하는 것이 좋습니다. <ol start="4"><li> 멀티 컬러 보정을 수행하는 하나의 스테인드 컨트롤이나 항체 캡처 보상 비즈를 사용하고 있습니다. </li></ol> 참고 : 보상 구슬의 사용이 권장되지만, 각 항체의 호환성을 보장해야합니다. 예를 들어, 보상 비즈 고립 된 세포가 적절한 하나의 얼룩 제어를 얻기 위해 요구되는 경우에 토끼 항체와 교차 반응하지 않을 수 있습니다. <ol start="5"><li> 수정 FMO 컨트롤에 따라 실험 게이트를 설정합니다. </li><li> 그 인구의 보급에 따라 이벤트의 해당 번호를 수집하고 실험 데이터를 기록하는 흐름 cytometer에 설정합니다. </li><li> 수출 FCS 데이터를 오프라인 분석을 위해 파일. 흐름 cytometr 가능한 많은 프로그램이 있습니다Y 데이터 분석. 우리는 Cytobank, investigatores 인터넷 액세스 18와 함께 모든 컴퓨터에서 그림을 저장하고 분석 데이터 및 생성 할 수있는 웹 기반의 플랫폼을 권장합니다. 또한, Cytobank 데이터 공개에 액세스 할 수 있도록하거나 공동 작업자에 대한 액세스를 제한 할 수있는 기능을 제공합니다. </li></ol>

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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=Characterizing+and+quantifying+leukocyte+populations+in+human+adipose+tissue:+impact+of+enzymatic+tissue+processing.">Characterizing and quantifying leukocyte populations in human adipose tissue: impact of enzymatic tissue processing.</a> Journal of Immunological Methods. 386, 50-59 (2012).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> J. Vis. Exp. (41), e1546 (2010).</li><li>Kotecha, N., Krutzik, P. O., Irish, J. 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H., 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=Angiogenic+role+of+LYVE-1-positive+macrophages+in+adipose+tissue.">Angiogenic role of LYVE-1-positive macrophages in adipose tissue.</a> Circulation Research. 100, e47-e57 (2007).</li><li>Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+compensation+for+flow+cytometry:+visualization+artifacts,+limitations,+and+caveats.">Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats.</a> Cytometry. 45, 194-205 (2001).</li></ol>

우리는 공개 아무것도 없어.

비만의 신진 대사 결과에서 면역 체계의 역할에 대한 관심의 증가는 AT의 면역 세포를 특성화 유동 세포 계측법의 광범위한 사용하게되었다. 정확한 프로토콜은 자신의 경험과 장비를 사용할 기준으로 실험실 사이에 다를 수 있지만, 중요한 단계는 콜라 소화, 차등 원심 분리하고, 세포 표면 항원 라벨 (가) 있습니다. 본 문서의 목적은 AT 및 / 또는 유동 세포 계측법을 수행하는 작업에 대한 경험을...

역사적으로, 지방 조직 (AT)는 에너지 균형에 대한 응답으로 신축 지질 스토리지의 불활성 기관으로 조회되었습니다. 우리는 지금 AT 적극적으로 직접 먹이 행동과 조직의 포도당 항상성에 영향을 미치는 호르몬의 숫자를 분비 동적 내분비 기관을 나타내는 것을 알 수 있습니다. 또한, 지난 10 년 동안 각막 혈관 분획 (SVF)뿐만 아니라 항상성에 그들의 공헌 AT에 거주하는 면역 세포의 다양한 인구에 대한 증가하는 감사가있다. 1964 1 Rodbell에 의해 설명 된 지방 세포와 SVF는 콜라게나 다이제스트를 사용하여 AT를 분리하는 기능은 차등 원심 분리에 의해 따랐다. 콜라게나 II는 대부분 지방 세포의 인슐린 수용체 1의 보수에 의한 지방 세포와 SVF 분리하는 데 사용됩니다. AT의 초기에, 효소 분획은 주로 adipo을 연구하기 위해 고용되었다신진 대사를 cyte 및 preadipocytes를 분리 할 수​​ 있습니다. 더 최근에, 흐름 cytometers의 광범위한 가용성과 상업적으로 이용 가능한 형광 - 복합 항체의 계속 증가와 함께이 기술은 면역 세포 AT의 특성을 촉진했다. AT 염증 면역 세포의 존재가 이전에이 설명되어 있었지만, 와이즈 버그 등으로 정액 논문. 그리고 쑤 하였다. 출판은 2003 년에 염증성 사이토 카인을 분비하고 AT 별 및 조직 인슐린 저항성 3,4와 연관 비만 식세포 AT의 축적 (현금 인출기)을 문서화하는 첫번째이었다. 이러한 관찰은 연구의 새로운 분야의 기초 최근에, &quot;immunometabolism,&quot;10 신조어로 제공하고 돌기 세포 6, 비만 세포 7, T 세포 8 등 다양한 면역 세포 인구를, 연루 연구에 의해 미행 당하고있다 -10, B 세포 11, NKT 세포 12, 호산구 13, 및 호중구 비만 관련 인슐린 저항의 개발에 14,15. 이 문서의 목적은 SVF AT의 세포를 분리하고 현금 인출기의 특성 및 유동 세포 계측법을 통해 T 세포 AT에 사용되는 콜라 다이제스트 기술에 대한 자세한 설명을 제공하는 것입니다. 이 프로토콜은 AT 마우스에 최적화되어 있지만, 시청자는 16 AT 인간이 기술의 최적화에 대한 광범위한 세부 정보를 제공하는 우수한 기사를 읽고 혜택을 누릴 수 있습니다. 이 문서의 대상은 제한된 경험 AT 마우스 작업 및 유동 세포 계측법을 수행하기에 수사관을 포함합니다. 시간과 자원을 가진 세포 수율과 생존의 균형을위한 몇 가지 실제적인 고려 사항은 면역 세포 인구 AT 특성화뿐만 아니라 최적의 유동 세포 계측법 컨트롤로 표시됩니다. 우리의 프로토콜뿐만 아니라, 독자 referr 있습니다적절한 컨트롤과 보상 17를 포함하는 유동 세포 계측법의 기술적 측면의 일부 훌륭한 토론 바수 등.의 최근 조브 기사에 에드.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS (no Ca or Mg) 10 x 500 ml</td>
 <td>Life Technologies</td>
 <td>14190-250</td>
 <td></td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Life Technologies</td>
 <td>D3571</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A2153-100G</td>
 <td></td>
 </tr>
 <tr>
 <td>collagenase</td>
 <td>Sigma</td>
 <td>C6885-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>propidium iodide solution</td>
 <td>Sigma</td>
 <td>P4864-10 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 20 microliter</td>
 <td>Rainin</td>
 <td>SS-L10</td>
 <td></td>
 </tr>
 <tr>
 <td>20 microliter filter tips</td>
 <td>Rainin</td>
 <td>SRL10F</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 250 microliter tips</td>
 <td>Rainin</td>
 <td>SS-L250</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter tips</td>
 <td>Rainin</td>
 <td>GPS-L1000</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter filter tips</td>
 <td>Rainin</td>
 <td>GP-L1000F</td>
 <td></td>
 </tr>
 <tr>
 <td>250 microliter Filter Tips</td>
 <td>Rainin</td>
 <td>SR-L200F</td>
 <td></td>
 </tr>
 <tr>
 <td>FC Block</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td></td>
 </tr>
 <tr>
 <td>fisher 100mn strainers</td>
 <td>Fisherbrand</td>
 <td>22-363-549</td>
 <td></td>
 </tr>
 <tr>
 <td>medium weigh dish</td>
 <td>Fisherbrand</td>
 <td>02-202B</td>
 <td></td>
 </tr>
 <tr>
 <td>aluminum foil</td>
 <td>Fisherbrand</td>
 <td>1213100</td>
 <td></td>
 </tr>
 <tr>
 <td>mincing scissors</td>
 <td>Fisherbrand</td>
 <td>089531B</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Fisherbrand</td>
 <td>2215365</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml conical tubes</td>
 <td>BD Falcon</td>
 <td>14-959-49A</td>
 <td></td>
 </tr>
 <tr>
 <td>filter top FACS tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml pipette case 200</td>
 <td>BD Falcon</td>
 <td>1367520</td>
 <td></td>
 </tr>
 <tr>
 <td>round bottom tubes</td>
 <td>BD Falcon</td>
 <td>352058</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml syringe</td>
 <td>BD Falcon</td>
 <td>309646</td>
 <td></td>
 </tr>
 <tr>
 <td>V Bottom Plates</td>
 <td>Costar</td>
 <td>07-200-107</td>
 <td></td>
 </tr>
 <tr>
 <td>transfer bulb pipette</td>
 <td>Thermo Scientific</td>
 <td>13-711-22</td>
 <td></td>
 </tr>
 <tr>
 <td>Shaker</td>
 <td>Thermo Scientific</td>
 <td>11 676 071</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive mat</td>
 <td>Thermo Scientific</td>
 <td>1368750</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Culture Centrifuge</td>
 <td>Sorvall</td>
 <td>75253839</td>
 <td></td>
 </tr>
 <tr>
 <td>Adapters</td>
 <td>Sorvall</td>
 <td>75003723</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD16/CD32</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td>Concentration: 0.5 - 1 &mu;g/ 106 cells</td>
 </tr>
 <tr>
 <td>Rat anti-mouse F4/80</td>
 <td>eBioscience</td>
 <td>17-4801</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 17-4321</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD11b</td>
 <td>eBioscience</td>
 <td>11-0112</td>
 <td>Fluorophore conjugate: FITC 
 Concentration: 0.5 &mu;g/ 106 cells 
 Isotype control catalog number: 11/1/4031</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse CD11c</td>
 <td>eBioscience</td>
 <td>12-0114</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: Dec-88</td>
 </tr>
 <tr>
 <td>Goat anti-mouse MGL1/2</td>
 <td>R&amp;D Systems</td>
 <td>FAB4297P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Goat anti-mouse CD206</td>
 <td>R&amp;D Systems</td>
 <td>FAB2535P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CCR2</td>
 <td>R&amp;D Systems</td>
 <td>FAB5538P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC013P</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse TCR&beta;</td>
 <td>BD Biosciences</td>
 <td>553174</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 553956</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>552877</td>
 <td>Fluorophore conjugate: PE-Cy7 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: 552784</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD4</td>
 <td>BD Biosciences</td>
 <td>557956</td>
 <td>Fluorophore conjugate: Alexa Fluor 700 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 557963</td>
 </tr>
 </tbody>
</table>
Table 1. List of Materials and Reagents.

isolation of adipose tissue immune cells

비만 설치류와 인간의 지방 조직의 증가 식세포 침윤 (AT)의 발견은 로컬 및 전신 인슐린 저항 면역 세포 공헌에 대한 관심 강화하게되었다. 엎드려서 비만 AT 다른 면역 세포 집단의 분리 및 정량 지금 immunometabolism 실험실에서 일반적으로 활용 기법, 아직 극도의주의를 얻을 데이터가 안정적이고 해석되도록 기질 혈관 세포의 분리 및 유동 세포 계측법 분석을 모두 수행해야 . 이 비디오에서 우리는, 말하다 소화와 면역 세포가 풍부한 기질 혈관 부분을 분리하는 방법을 보여줍니다. 그 후, 우리는 항체 라벨 식세포와 T 림프구 방법과 유동 세포 계측법 실험에서 그들에 제대로 게이트하는 방법을 보여줍니다. 저지방 먹이 엎드려서 고지방 먹이 비만 쥐에서 대표 유동 세포 계측법 플롯이 제공됩니다. 이 분석의 중요한 요소는 어떻게 항체를 사용하는 것입니다대 식세포 AT 자연 autofluorescent중인 채널뿐만 아니라, 적절한 보상 컨트롤의 사용 형광 없습니다.

차등 원심 분리 한 다음 AT의 콜라 소화 저지방 (지방에서 10 % 킬로 칼로리) 또는 높은 지방 (지방 60 %의 킬로 칼로리) 다이어트 (LFD 및 HFD을 공급 수컷 C57BL/6J 마우스의 부고환 지방 패드에서 SVF를 분리하는 데 사용되었다 각각) 16 주. SVF의 세포는 다음 FACS 분석을 통해 가능한 ATM 기기 (그림 1)과 T 세포 (그림 2) AT 비율을 정량화하는 형광 - 복합 일차 항체로 표지 하였다. 빛?...

Watch this Scientific Journal Video about Isolation of Adipose Tissue Immune Cells at JoVE.com

Isolation of Adipose Tissue Immune Cells

지방 조직 (AT)는 강력한 면역 세포 활성화와 상호 작용의 사이트입니다. 면역 시스템의 거의 모든 세포는 AT의 존재와 그 비율은 비만에 의해 변경됩니다. 면역 세포 인구 AT의 적절한 분리, 정량 및 특성은 immunometabolic 질병에서 자신의 역할을 이해하는 데 중요하다.

지방 조직 면역 세포의 분리

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

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JoVE Journal

Immunology and Infection

35.8K 조회수.  Vanderbilt University School of Medicine. 지방 조직 (AT)는 강력한 면역 세포 활성화와 상호 작용의 사이트입니다. 면역 시스템의 거의 모든 세포는 AT의 존재와 그 비율은 비만에 의해 변경됩니다. 면역 세포 인구 AT의 적절한 분리, 정량 및 특성은 immunometabolic 질병에서 자신의 역할을 이해하는 데 중요하다.

비디오: Isolation of Adipose Tissue Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

연구

면역학 및 감염병학

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

유동세포계측법에 의해 피해를 입은 척수 조직에서 면역 세포의 양적 평가 : 세포 정제 방법에 대한 소설을 사용

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

셀을 별 모양으로 일곱 단계

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Murine Aortas 이내에 면역 세포의 흐름 Cytometry 분석

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

심장 기능 면역 세포의 분리

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

신장 면역 세포의 신뢰성 및 고효율 추출

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

절연, 확장, 및 Adipogenic CD34 + CD31 + 인간의 Omental 및 피하 지방 조직에서 내 피 세포의 유도

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...