Name: multicolor flow cytometry analyses of cellular immune response in rhesus macaques
Uploaded: 2010-04-22T17:35:00
Description: Watch this Scientific Journal Video about Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques at JoVE.com

普拉莫德Nehete博士和夫人Bharti公司Nehete猕猴血液和PBMC样品;部分支持这项工作是由美国国立卫生研究院授予的AI 46969资金，所有的细胞培养基，由中央媒体实验室，这是由美国国立卫生研究院授予CA资助16672。

总额以及记忆T细胞子集的表型和功能的详细分析的程序可分为四个部分：1）细胞的制备及抗原刺激，2）表面标志物染色，3）细胞内细胞因子染色和4）流式细胞仪分析。 1。细胞的制备及抗原刺激<ol><li>既新鲜分离以及冷冻保存外周血单个核细胞（PBMC）的，在此协议使用。肝素或枸橼酸恒河猴静脉血液样本中分离的外周血单核细胞密度梯度沉淀，使用聚蔗糖- Hypaque（Histopaq...

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	<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>

解决方案和缓冲器<ul><li>聚蔗糖- Hypaque（Histopaquen - 1077，Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li> RPMI - 1640培养基（Hyclone公司实验室，洛根，UT） </li><li>胎牛血清（FBS）（Hyclone公司实验室，洛根，UT） </li><li> L -谷氨酰胺（Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>青霉素/链霉素（Invitrogen公司;卡尔斯巴德，CA） </li><li>台盼蓝试剂（Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>的50 mM的Tris，PH值8.6，过滤和储存在4 ° C </li><li>贝科的PBS（DPBS，Ca2/Mg2-free; Life技术，马里兰州洛克维尔） </li><li>流洗涤缓冲液：含2％FBS DPBS补充，储存于4 ° C </li><li> BD Cytofix / Cytoperm固定/通透解决方案套件（BD Biosciences公司;加利福尼亚州圣何塞） </li></ul>激活剂<ul><li>佛波醇-12 - 肉豆蔻13醋酸乙烯酯（PMA）（Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>离子霉素（Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>共刺激表达CD49d单克隆抗体，克隆9F10（BD Biosciences公司;加利福尼亚州圣何塞） </li><li>亲和纯化抗体的F（ab）2片段的羊抗鼠IgG（H + L）（克尔凯郭尔和佩里实验室，马里兰州盖瑟斯堡） </li><li>特定的激活剂可以包括肽池，单肽，或整个蛋白质</li></ul>细胞因子的分泌抑制剂<ul><li> Brefedin一个（博鳌亚洲论坛，Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>高尔基的插头，可交替使用（BD Biosciences公司;加利福尼亚州圣何塞） </li></ul>抗体<ul><li>活/死可以解决旱厕荧光染色试剂盒（Invitrogen公司;卡尔斯巴德，CA） </li><li> CD3 + PE - Cy7（SP34 - 2），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li> CD4太平洋蓝（OKT4），（eBiosciences（加利福尼亚州圣迭戈） </li><li> CD8 + Alexa700（RPA - T8），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li> CD28 PerCP cy5.5（L293），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li> CD95的APC（DX2），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li>干扰素- FITC（B27），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li> IL - 2的PE（MQ1 - 17H12），（BD Biosciences公司;加利福尼亚州圣何塞） </li></ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

猕猴模型是目前最好的艾滋病毒和艾滋病的模型，了解其发病机制，以及疫苗和疗法的发展<sup&gt; 1,2,3</sup&gt;。在这里，我们描述了一个详细的表型和功能分析的细胞免疫反应，特别是细胞内的CD4细胞因子生产+和CD8 + T细胞以及个人记忆亚群的方法。我们精确的定量和定性的措施，为生产干扰素γ（INF -γ）和白细胞介素（IL）-2获得的CD4 +和CD8 + T细胞从PMA加离子霉素（PMA + I）刺激的恒河猴PBMC的。细胞因子在记忆细胞不同亚群的不同。此外，该协议为我们提供的灵敏度，证明即使是轻微的组分抗原特异性CD4 +和CD8 + T细胞内的PBMC样本在我们的实验室开发的HIV包膜肽鸡尾酒疫苗免疫恒河猴的子集。多色流式细胞术是一种强大的工具，准确地识别不同人群的T细胞<sup&gt; 4,5</sup&gt;与细胞因子产生能力<sup&gt; 6</sup&gt;非特异性或抗原特异性刺激以下<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

我们展示了总额以及记忆CD4 +和CD8 + T细胞在猕猴，艾滋病毒/艾滋病疫苗研究的理想模型的子集的详细的表型和功能特性的多色流式细胞仪的实用。

多色流流式细胞仪分析细胞在恒河猴的免疫应答

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 ...

Research

JoVE Journal

Immunology and Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

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

科研

免疫与感染

Seven Steps to Stellate Cells

星状细胞的七个步骤

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

流式细胞仪分析小鼠主动脉内流的免疫细胞

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

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

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

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

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

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

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

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

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

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

猴免疫缺陷病毒特异性CD8分析<sup&gt; +</sup&gt; T细胞猕猴由肽MHC-I四聚体染色

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

影像性流式细胞仪对人体免疫突触的定性和定量分析

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

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

猕猴免疫缺陷病毒感染 CD4+ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...