多色流流式细胞仪分析细胞在恒河猴的免疫应答
Summary
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。
Abstract
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> 1,2,3</sup>。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> 4,5</sup>与细胞因子产生能力<sup> 6</sup>非特异性或抗原特异性刺激以下<sup> 5,7</sup>。
Protocol
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…
Acknowledgements
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。
Materials
Solution and Buffers
- Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO)
- RPMI-1640 medium (HyClone laboratories, Logan, UT)
- Fetal Bovine Serum (FBS), (HyClone laboratories, Logan, UT)
- L-glutamine (Sigma-Aldrich; St. Louis, MO)
- Penicillin/streptomycin (Invitrogen; Carlsbad, CA)
- Trypan blue reagent (Sigma-Aldrich; St. Louis, MO)
- 50 mM Tris, PH 8.6; filtered and store at 4°C
- Dulbecco’s PBS (DPBS, Ca2/Mg2-free; Life technologies, Rockville, MD)
- Flow wash buffer: DPBS supplement with 2% FBS, store at 4°C
- BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences; San Jose, CA)
Activation agents
- Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO)
- Ionomycin (Sigma-Aldrich, St. Louis, MO)
- Costimulatory mAb CD49d, clone 9F10 (BD Biosciences; San Jose, CA)
- Affinity purified antibody F (ab )2 fragments of Goat Anti-Mouse IgG (H + L) (Kierkegaard and Perry Laboratory, Gaithersburg, MD)
- Specific activation agents could include peptide pools, single peptide, or whole protein
Cytokine secretion inhibitors
- Brefedin A (BFA, Sigma-Aldrich, St. Louis, MO)
- Golgi Plug can be used alternatively (BD Biosciences; San Jose, CA)
Antibodies
- Live/dead fixable aqua fluorescent stain kit (Invitrogen; Carlsbad, CA)
- CD3 PE-Cy7 (SP34-2), (BD Biosciences; San Jose, CA)
- CD4 pacific blue (OKT4), (eBiosciences (San Diego, CA)
- CD8 Alexa700 (RPA-T8), (BD Biosciences; San Jose, CA)
- CD28 PerCP-cy5.5 (L293), (BD Biosciences; San Jose, CA)
- CD95 APC (DX2), (BD Biosciences; San Jose, CA)
- IFN- FITC (B27), (BD Biosciences; San Jose, CA)
- IL-2 PE (MQ1-17H12), (BD Biosciences; San Jose, CA)
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Tags
Multicolor Flow CytometryCellular Immune ResponseRhesus MacaquesHIV-AIDS ModelPathogenesisVaccinesTherapeuticsPhenotypic AnalysisFunctional AnalysisIntracellular Cytokine ProductionCD4+ T CellsCD8+ T CellsMemory SubsetsInterferon Gamma (INF-)Interleukin (IL)-2PMA Plus Ionomycin (PMA+I)Cytokine ProfilesMemory Cells SubsetsAntigen-specific CD4+ T CellsAntigen-specific CD8+ T CellsPBMC SamplesHIV Envelope Peptide Cocktail Vaccine
Citer Cet Article
He, H., Courtney, A. N., Wieder, E., Sastry, K. J. Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques. J. Vis. Exp. (38), e1743, doi:10.3791/1743 (2010).