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

Dr. Pramod Nehete and Mrs. Bharti Nehete for macaque blood and PBMC samples; This work was partially supported by funds from NIH grant AI 46969, and all the cell culture media were produced by the Central Media Laboratory, which is funded by NIH grant CA 16672.

The procedures for the detailed phenotypic and functional analyses of total as well as memory subsets of T cells can be divided into four parts: 1) Cell preparation and antigenic stimulation, 2) Surface marker staining, 3) Intracellular cytokine staining and 4) Flow cytometry analyses.
1. Cell preparation and antigenic stimulation
<ol>
	<li>Both freshly isolated as well as cryo-preserved peripheral blood mononuclear cells (PBMC) were used in this protocol. The PBMC were isolated from heparin...

<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>

Solution and Buffers
<ul>
 <li>Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO)</li>
 <li>RPMI-1640 medium (HyClone laboratories, Logan, UT)</li>
 <li>Fetal Bovine Serum (FBS), (HyClone laboratories, Logan, UT)</li>
 <li>L-glutamine (Sigma-Aldrich; St. Louis, MO)</li>
 <li>Penicillin/streptomycin (Invitrogen; Carlsbad, CA)</li>
 <li>Trypan blue reagent (Sigma-Aldrich; St. Louis, MO)</li>
 <li>50 mM Tris, PH 8.6; filtered and store at 4&deg;C</li>
 <li>Dulbecco's PBS (DPBS, Ca2/Mg2-free; Life technologies, Rockville, MD)</li>
 <li>Flow wash buffer: DPBS supplement with 2% FBS, store at 4&deg;C</li>
 <li>BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences; San Jose, CA)</li>
</ul>
Activation agents
<ul>
 <li>Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) </li>
 <li>Ionomycin (Sigma-Aldrich, St. Louis, MO)</li>
 <li>Costimulatory mAb CD49d, clone 9F10 (BD Biosciences; San Jose, CA) </li>
 <li>Affinity purified antibody F (ab )2 fragments of Goat Anti-Mouse IgG (H + L) (Kierkegaard and Perry Laboratory, Gaithersburg, MD)</li>
 <li>Specific activation agents could include peptide pools, single peptide, or whole protein</li>
</ul>
Cytokine secretion inhibitors
<ul>
 <li>Brefedin A (BFA, Sigma-Aldrich, St. Louis, MO)</li>
 <li>Golgi Plug can be used alternatively (BD Biosciences; San Jose, CA)</li>
</ul>
Antibodies
<ul>
 <li>Live/dead fixable aqua fluorescent stain kit (Invitrogen; Carlsbad, CA)</li>
 <li>CD3 PE-Cy7 (SP34-2), (BD Biosciences; San Jose, CA)</li>
 <li>CD4 pacific blue (OKT4), (eBiosciences (San Diego, CA)</li>
 <li>CD8 Alexa700 (RPA-T8), (BD Biosciences; San Jose, CA) </li>
 <li>CD28 PerCP-cy5.5 (L293), (BD Biosciences; San Jose, CA)</li>
 <li>CD95 APC (DX2), (BD Biosciences; San Jose, CA)</li>
 <li>IFN- FITC (B27), (BD Biosciences; San Jose, CA)</li>
 <li>IL-2 PE (MQ1-17H12), (BD Biosciences; San Jose, CA)</li>
</ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

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 of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

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

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

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 ...

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