짧은 꼬리 원숭이 혈액과 PBMC 샘플 박사 프라 모드 Nehete 부부 바티 Nehete,이 작품은 부분적으로 NIH 부여 AI 46,969의 자금에 의해 지원되었고, 모든 세포 배양 매체는 중앙 미디어 연구소에 의해 생산되었다, NIH 부여 CA에 의해 자금 지원됩니다 16672.

1) 셀 준비와 antigenic의 자극, 2) 표면 마커 얼룩, 3) 세포 시토킨 얼룩과 4) 유동세포계측법 : 총뿐만 아니라 T 세포의 메모리 하위 집합의 세부 phenotypic 및 기능 분석에 대한 절차는 네 부분으로 나눌 수 분석합니다. 1. 세포 준비와 antigenic의 자극 <ol><li> 두 신선하게 격리뿐만 아니라 cryo 보존된 말초혈 mononuclear 세포 (PBMC)가이 프로토콜에 사용되었습니다. PBMC는 Ficoll - Hypaque (Histopaquen - 1077, 시그마 - 알드리치, ST. 루이스, MO)를 사용하여 밀도 기울기 침강에 의해 붉은 털원숭이 짧은 꼬리 원숭이의 heparinized 또는 citrated 정맥의 혈액 샘플로부터 격리되었다. PBMC의 Aliquots는 90% FCS에 냉동 저장 및 액체 질소의 10 % DMSO되었습니다. </li><li> cryo - 보존된 PBMC를 사용하는 경우, 냉동 PBMC의 유리병은 액체 질소에서 제거되었으며 신속하게 냉동 매체와를 제거하는 RPMI - 1640 (HyClone 실험실, 로건, UT)로 씻어, 부드럽게 혼합, 37 C의 물을 욕조에 해동 전체 미디어 다시 정지 [CM, 10 %로 RPMI - 1640 보충 열 inactivated FCS (HyClone 연구소), 2mM L - 글루타민 (시그마 - 알드리치), 100U/ml 페니실린 / 스트렙토 마이신 (Invitrogen), 그리고 6 교양 - 잘 조직 배양 플레이트 37 ° 하룻밤 humidified 5 % CO 2 분위기에서 C. 다음날 아침은 생균 카운트는 trypan - 청색 색소 배제 방식에 의해 결정 및 CM에서 다시 정지. </li><li> 세포 시토킨 분석하여 다음 불특정 대 항원에 특정한 자극에 대해 다르​​게 대우했다 : (A) phorbol 12 - myristate와 비 특정 자극에 대한 13 아세테이트 (PMA)와 Ionomycin (I) (시그마 - 알드리치, 성 루이, MO), 0.1ml의 볼륨에있는 세포의 aliquots (1.0 X 10 6 세포 / 음)은 96 잘 조직 배양 플레이트 (BD Biosciences, 프랭클린 호수, 뉴저지)의 개별 우물에서 도금했다. PMA와 Ionomycin는 각각 50ng/ml과 500ng/ml의 최종 농도에서 사용되었습니다. 96 - 웰 조직 배양 플레이트에 대한 최종 합계 볼륨 0.2ml/well입니다 (b)는 항원 특정 자극 들어, 볼륨 1.0 ML 1.0 X 10 6 세포는 24 잘 조직 문화 판의 개별 웰스 (BD Biosciences에서 도금했다 이전 수정 9에서 설명하는 것처럼 우물은 사전 처리되었습니다 프랭클린 호수, NJ). 간단히, 24 잘 조직 문화 플레이트는 4 50mM 트리스 솔루션 2.5 μg / ML / 잘 염소 안티 - 마우스 IgG (H + L) (Kierkegaard 및 페리 연구소, 게이 더 스버그, MD) (PH 8.6)로 코팅했다 ° C 하룻밤. 접시가 멸균 PBS와 공동 stimulatory mAb CD49d, 클론 9F10 (BD Biosciences, 산호세, CA)를 두 번 씻은되었습니다 다음날 아침이 37에 한 시간 동안 잠복기 10 μg / ML에 추가 / 잘 따라했다 ° C.가 부화 후, 접시는 실온에서 멸균 PBS로 두 번 씻어했다. 여섯 HIV 봉투 펩티드 8 칵테일을 포함하여 관심이 항원은 (각 펩타이드의 10μg/ml의 최종 농도에 추가되었습니다. 혼자 CM의 세포로 추가 우물은 대조군으로 준비되었습니다. 24의 각 잘에 대한 최종 합계 볼륨 - 잘 조직 배양 플레이트 1.0 ML되었습니다. </li><li> 세포는 37 6 시간 동안 교양되었습니다 ° C humidified 5 % CO 2 분위기 인치 Brefeldin이 (시그마 - 알드리치, 세인트 루이스, MO) 자극의 마지막 4.5 시간 10μg/ml의 문화에 추가되었습니다. 이후, 전지 5 ML의 폴리 프로필렌 튜브로 전송과 함께 세탁했다 추운 (4 ° C) 유동 세척 버퍼 (Dulbecco의 PBS (DPBS, CA 2 / MG 2 무료, 함께 생활 기술, 락빌, MD) 2퍼센트 열 inactivated FCS), 그리고 다양한 형광 라벨 항체와 얼룩을위한 처리. </li></ol> 2. 얼룩 Immunofluorescence 표면 마커 <ol><li> 자극 세포 처음 4 1ul 죽은 / 라이브 고칠수 아쿠아 형광 반응 염료 물들일되었습니다 ° 30 분 동안 어둠 속에 C, 차가운 흐름 세척 버퍼와 한번 세탁. </li><li> 그런 다음, 다음 적절하게 titrated 형광 - 표시 단클론 항체가 세포에 추가되었습니다 방지 인 경우에는 3 번 CD에 들어 PE - Cy7 (SP34 - 2), 안티 - CD8 Alexa700 (RPA - T8), 안티 CD28 PerCP - cy5.5 (L293) , 안티 CD95 APC (DX2) 및 안티 CD4 퍼시픽 블루 (OKT4), (샌디에고, CA) eBioscience에서 CD4 제외 BD Biosciences (프랭클린 호수, NJ)에서, 퍼시픽 블루 (OKT4). 세포는 어두운 4 30 분 ° C에 대한 incubated되었습니다. </li><li> 각 실험에 대한 보상 컨트롤과 형광 빼기 1 (FMO) 모두 10,11가 활용되었다 제어합니다. 보정 컨트롤은 다른 메이커에 얼룩 각의 다른 일의 coeffecients을 결정하기 위해 이용되었다. 보상 컨트롤, 원숭이 중 하나에서 세포 suspensions를 포함하는 튜브의 전체 집합은 단일 색상의 얼룩으로 개별적으로 형광 복합 mAb 물들일되었습니다. FMO는 모든 시약하지만 관심 중 하나를 포함하는 여러 가지 빛깔의 얼룩의 조합이며 긍정 사이의 경계를 결정하는 데 사용됩니다자동 형광 레벨 및 데이터를 복제하여 ND 부정적인 인구는 완전히 스테인드 샘플에 존재. </li><li> 스테인드 세포는 다음 차가운 흐름 세척 버퍼로 세탁하고, 고정 / permeabilization 솔루션 (BD Biosciences, 산호세, CA)에서 해결되었습니다 (이 단계에서 세포가 4 밤새 저장할 수 있습니다 ° C를 어둠 속에서) 적어도 10 분 동안 세포내 시토킨 얼룩을 수행하기 전에. </li></ol> 3. 세포내 시토킨 염색법 <ol><li> 고정 전지는 유동 세척 버퍼로 씻어되었고, 다음 펌 / (BD Biosciences, 산호세, CA) 버퍼를 씻으는 제조 업체의 지시에 따라 추가되었습니다. 간단히, 세포는 1X 펌 / 4 10 분 버퍼를 씻으 ° 어둠 속에 C의 0.1ml에 incubated했다. </li><li> 이에 따라 전지와 incubated 있던 적절 titrated FITC - 라벨 방지 IFN - γ (B27)와 PE - 라벨 4 60 분 반 IL - 2 (MQ1 - 17H12) · 어둠 속에 C. </li><li> 그러면 세포는 permeabilization 솔루션과 두 번 더 씻어되었습니다 </li><li> 최종 세척 후, 세포가 DPBS의 1 % paraformaldehyde에 다시 정지하고 24 시간 이내에 유동세포계측법 분석의 대상이되었다. </li></ol> 4. 유동세포계측법 분석 <ol><li> 또는 청록색 ADP (Dako, 카핀테리아, CA), 스테인드 세포는 LSR II 흐름 cytometer (산호세, CA BD Biosciences)에 인수했다. 외과 데이터는 FlowJo 소프트웨어 (트리 스타, 애쉬 랜드, OR)를 사용하여 분석되었다. </li><li> 그림. 1 대표적인 동물에서 다른 T 세포의 하위 집합의 분석 활용 게이팅 구조를 보여줍니다. lymphocytes 앞으로 분산형 (FSC) 대 사이드 분산형 (SSC)를 통해 첫번째 문이 있었고, 다음 라이브 lymphocytes는 SSC와 아쿠아 부정적인 인구를 기반으로 식별되었습니다. 인 경우에는 3 번 CD 내의 T 세포는 다음 적극적으로 CD4의 감지 + CD8 - (CD4 + T 세포)에 의해 다음 인 경우에는 3 번 CD에 들어 표현으로 식별 및 CD4 - CD8 + (CD8 + T 세포)되었습니다 인구 + T 세포 인구. CD4 +와 CD8 + T 세포는 더 이상 순진 (TN, CD28 + CD95 -), 중앙 메모리 (TCM, CD28 + CD95 +) 및 효과기 메모리 (TEM, CD28 - CD95 +) 세포처럼 CD28과 CD95 표현에 근거하여 서로 다른 하위 집합으로 구분되었다 문학 5, 10에 설명되어. </li><li> 그림. CD4 +와 CD8 + T 세포의 서로 다른 하위 집합은 PMA와 ionomycin (PMA + I)과 자극에 대한 응답으로 시토킨 생산의 관점에서 기능적 용량 (INF - γ 및 / 또는 IL - 2)에 대한 평가가 얼마나이 표시됩니다. </li></ol> 5. 대표 외과 데이터 붉은 털원숭이 짧은 꼬리 원숭이의 PBMC 구성은 외과 분석을 사용하여 결정되었다. 그림 1은 대표적인 동물에서 다른 T 세포의 하위 집합의 분석 활용 게이팅 구조를 보여줍니다. lymphocytes 앞으로 분산형 (FSC) 대 사이드 분산형 (SSC)를 통해 첫번째 문이 있었고, 다음 라이브 lymphocytes는 SSC와 아쿠아 부정적인 인구를 기반으로 식별되었습니다. T 세포는 다음 인 경우에는 3 번 CD에 들어 표현식에 의해 확인되었습니다. 인 경우에는 3 번 CD에 들어 내에서 CD4 + CD8 - (CD4 + T 세포)와 CD4 - CD8 + (CD8 + T 세포) 인구는 + T 세포 인구도 결정됩니다. 그림 2에 표시된 CD4 +와 CD8 + T 세포는 더 이상 순진 (TN, CD28 + CD95 -), 중앙 메모리 (TCM, CD28 + CD95 +) 및 효과기 메모리 (TEM로 CD28과 CD95 표현에 근거하여 서로 다른 하위 집합으로 구분되었다 CD28 - CD95 +) 세포. 그림 2A는 INF - γ를 보여줍 unstimulated 및 PMA / ionomycin 자극 CD4 + T 세포, 그리고 하위 집합, 그리고 그림 2B에서 IL - 2 생산 INF - γ와 unstimulated 및 PMA / ionomycin가 CD8을 자극 + T 세포에서 IL - 2 생산을 보여줍니다, 그리고 집합 뚜렷한 시토킨 프로파일 각 하위 집합에 자극에 의​​해 elicited합니다. <img src="/files/ftp_upload/1743/1743fig1tmb.jpg" alt="그림 1" border="0" /> 그림 1. 게이팅 전략은 붉은 털원숭이 macqaues의 PBMC 구성의 흐름 cytometric 분석에 사용. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1743/1743fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다. <img src="/files/ftp_upload/1743/1743fig2atmb.jpg" alt="그림 2A" border="0" /> <img src="/files/ftp_upload/1743/1743fig2btmb.jpg" alt="그림 2B" border="0" /> 그림 2. (A) 총 CD4 + T 세포와 하위 집합.의 시토킨 생산 프로파일 총 CD8 + T 세포와 하위 집합의 (B) 시토킨 생산 프로파일. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2a.jpg">여기를 클릭하십시오</a> 큰 2A의 버전을보고 <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2b.jpg">여기 또는</a> 그림 2B의 큰 버전에 대한.

<ol>
	<li>Ogg, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+HIV-1-specific+cytotoxic+T+lymphocytes+and+plasma+load+of+viral+RNA.">Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.</a> Science. 279, 2103-2106 (1998).</li><li>Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M., Oldstone, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-specific+CD8++cytotoxic+T-lymphocyte+activity+associated+with+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+infection.">Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.</a> J Virol. 68, 6103-6110 (1994).</li><li>Koup, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+association+of+cellular+immune+responses+with+the+initial+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+syndrome.">Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.</a> J Virol. 68, 4650-4655 (1994).</li><li>Walker, J. M., Maecker, H. T., Maino, V. C., Picker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+flow+cytometric+analysis+in+SIV-infected+rhesus+macaque.">Multicolor flow cytometric analysis in SIV-infected rhesus macaque.</a> Methods Cell Biol. 75, 535-557 (2004).</li><li>Pitcher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+T+cell+memory+in+rhesus+macaque.">Development and homeostasis of T cell memory in rhesus macaque.</a> J Immunol. 168, 29-43 (2002).</li><li>Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracellular+cytokines+by+flow+cytometry.">Detection of intracellular cytokines by flow cytometry.</a> J Immunol Methods. 159, 197-207 (1993).</li><li>Keeney, T. S., Nomura, L. E., Maecker, H. T., Sastry, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+macaque+whole+blood+for+antigen-specific+intracellular+cytokine+production+by+T+lymphocytes.">Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes.</a> J Med Primatol. 32, 23-30 (2003).</li><li>Gauduin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intracellular+cytokine+staining+for+the+quantitation+of+antigen-specific+CD4++T+cell+responses+in+rhesus+macaques.">Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques.</a> J Immunol Methods. 288, 61-79 (2004).</li><li>Baumgarth, N., Roederer, 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=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243, 77-97 (2000).</li><li>De Rosa, S. C., Herzenberg, L. A., Roederer, M. 1. 1. -. c. o. l. o. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13-parameter+flow+cytometry:+identification+of+human+naive+T+cells+by+phenotype,+function,+and+T-cell+receptor+diversity.">13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.</a> Nat Med. 7, 245-248 (2001).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+fluorescence+compensation+and+visualization+of+FACS+data.">New approaches to fluorescence compensation and visualization of FACS data.</a> Clin Immunol. 110, 277-283 (2004).</li></ol>

솔루션 및 버퍼 <ul><li> Ficoll - Hypaque (Histopaquen - 1077, 시그마 - 알드리치, 세인트 루이스, MO) </li><li> RPMI - 1640 배지 (HyClone 실험실, 로건, UT) </li><li> 태아 소 혈청 (FBS), (HyClone 실험실, 로건, UT) </li><li> L - 글루타민 (시그마 - 알드리치, 세인트 루이스, MO) </li><li> 페니실린은 / 스트렙토 마이신 (Invitrogen, 칼스 배드, CA) </li><li> Trypan 파란색 시약 (시그마 - 알드리치, 세인트 루이스, MO) </li><li> 50 MM 트리스, PH 8.6, 4 필터링과 가게 ° C </li><li> Dulbecco의 PBS (DPBS, Ca2/Mg2-free, 생명 기술, 락빌, MD) </li><li> 흐름 세척 버퍼 : 2% FBS와 DPBS 보완, 4 저장 ° C </li><li> BD Cytofix / Cytoperm 고정 / Permeabilization 솔루션 키트 (BD Biosciences, 산호세, CA) </li></ul> 활성화 요원 <ul><li> Phorbol 12 - myristate 13 - 아세테이트 (PMA) (시그마 - 알드리치, 세인트 루이스, MO) </li><li> Ionomycin (시그마 - 알드리치, 세인트 루이스, MO) </li><li> Costimulatory mAb CD49d, 클론 9F10 (BD Biosciences, 산호세, CA) </li><li> 친화력 정화 항체 F (AB) 염소 방지 마우스 IgG (H + L) 2 조각 (Kierkegaard 및 페리 연구소, 게이 더 스버그, MD) </li><li> 특정 인증 요원들은 펩타이드 수영장, 단일 펩티드, 또는 전체 단백질을 포함시킬 수 </li></ul> 시토킨 분비 억제제 <ul><li> Brefedin A (BFA, 시그마 - 알드리치, 세인트 루이스, MO) </li><li> 잠돌군 Zz 플러그는 (; 산호세, CA BD Biosciences) 또는 사용할 수 있습니다 </li></ul> 항체 <ul><li> 죽은 / 라이브 고칠수 아쿠아 형광 얼룩 키트 (Invitrogen, 칼스 배드, CA) </li><li> 인 경우에는 3 번 CD에 들어 PE - Cy7 (SP34 - 2), (BD Biosciences, 산호세, CA) </li><li> CD4 태평양 블루 (OKT4), (eBiosciences (샌디에고, CA) </li><li> CD8 Alexa700 (RPA - T8), (BD Biosciences, 산호세, CA) </li><li> CD28 PerCP - cy5.5 (L293), (BD Biosciences, 산호세, CA) </li><li> CD95 APC (DX2), (BD Biosciences, 산호세, CA) </li><li> IFN - FITC (B27), (BD Biosciences, 산호세, CA) </li><li> IL - 2 PE (MQ1 - 17H12), (BD Biosciences, 산호세, CA) </li></ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

붉은 털원숭이 짧은 꼬리 원숭이 모델은 현재 백신 및 치료제의 개발을위한 최상의 사용할 수있는 pathogenesis을 이해와 관련하여 HIV - AIDS에 대한 모델뿐 아니라<sup&gt; 1,2,3</sup&gt;. 여기, 우리는 세포 면역 반응에 대한 자세한 phenotypic 및 기능 분석, CD4 세포에 의해 구체적으로 시토킨 생산 +와 CD8 + T 세포뿐만 아니라 개별 메모리 하위 집합에 대한 방법을 설명합니다. 우리는 모두 CD4에서 인터페론 감마의 생산에 대한 정확한 양적 및 질적 조치 (INF -)와 인터루킨 (IL) -2를 취득 +와 CD8 + T PMA 플러스 ionomycin (PMA + I)를 자극 붉은 털원숭이 짧은 꼬리 원숭이 PBMC에서 세포. 시토킨 프로파일 메모리 세포의 서로 다른 하위 집합에서 다른되었습니다. 또한,이 프로토콜은 항원 특정 CD4의도 작은 분수를 입증하기 위해 우리에게 감도를 제공하는 +와 CD8 + 우리 연구실에서 개발된 HIV 봉투 펩타이드 칵테일 백신과 예방 접종을 붉은 털원숭이 짧은 꼬리 원숭이에서 PBMC 샘플 내의 T 세포 집합. 여러 가지 빛깔의의 유동세포계측법 기술 정확하게 T 세포의 다른 인구를 식별하는 강력한 도구입니다<sup&gt; 4,5</sup&gt; 시토킨 생산 기능이있는<sup&gt; 6</sup&gt; 불특정 또는 특정 항원 자극에 따라<sup&gt; 5,7</sup&gt;.

Watch this Scientific Journal Video about Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques at JoVE.com

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

우리는 총뿐만 아니라 붉은 털원숭이 짧은 꼬리 원숭이의 CD4 +와 CD8 + T 세포, HIV / AIDS 백신 연구를위한 이상적인 모델의 메모리 하위 집합에 대한 자세한 phenotypic와 기능 특성에 대한 여러 가지 빛깔의의 유동세포계측법의 유틸리티를 보여줍니다.

붉은 털원숭이 짧은 꼬리 원숭이의 세포 면역 반응의 여러 가지 빛깔의의 유동세포계측법의 분석

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

multicolor-flow-cytometry-analyses-cellular-immune-response-rhesus

Research

JoVE Journal

Immunology and Infection

21.0K 조회수.  MD Anderson Cancer Center - University of Texas. 우리는 총뿐만 아니라 붉은 털원숭이 짧은 꼬리 원숭이의 CD4 +와 CD8 + T 세포, HIV / AIDS 백신 연구를위한 이상적인 모델의 메모리 하위 집합에 대한 자세한 phenotypic와 기능 특성에 대한 여러 가지 빛깔의의 유동세포계측법의 유틸리티를 보여줍니다.

비디오: Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

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

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 ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

사람의 체외에서</em면역 불균형의 초기 국가의 인정을위한 심사 도구를> 억제

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

유동 세포 계측법에 의해 주요 면역 세포 모집단과 형광 나노 입자의 상호 작용의 검출

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

유인원 면역 결핍 바이러스 특정 CD8 분석<sup&gt; +</sup&gt; 펩티드 - MHC-I 테트라 머 염색에 의해 붉은 털 원숭이의 T 세포

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

Cytometry 이미징 사용 하 여 인간의 시스템에 면역 시 냅 스의 질적 및 양적 분석

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.