Multiparameter Flow Cytometric Analysis of T Cell Subsets

Isolated cells were labeled with a combination of CD3-Cy7APC, CD4-PB, CD28-Cy5-PE, CD95-FITC, and CD8-Alexafluor700 and analyzed by flow cytometry using procedures described previously [10 (link), 12 (link), 18 (link), 20 (link)–22 (link)]. All the antibodies were obtained from BD Biosciences (San Diego, CA) and titrated using rhesus macaque PBMC. Labeled cells were fixed with 0.5% paraformaldehyde and analyzed using a Becton Dickinson LSR II flow cytometer. For analysis of SIV-gag DNA in CD4⁺ T cells, cells were labeled as above and CD4⁺ T cells were sorted using a BD FACS Aria sorter.
IL-17 production in CD4⁺ T cells was determined after short-term stimulation with 10 ng/mL of PMA (phorbol myristate acetate; Sigma-Aldrich, Saint Louis, MO) and 500 ng/mL of Ionomycin (Sigma-Aldrich, Saint Louis, MO) in the presence of Brefeldin-A (BD Biosciences) for 4 hours. After stimulation, cells were labeled with anti-CD3-Cy7APC, CD8-Alexa-700, and CD4-PE. Cells were fixed in Cytofix/perm buffer (BD Biosciences) and labeled with anti-IL-17-APC (e-Biosciences). Labeled cells were fixed in 0.5% paraformaldehyde and analyzed using a Becton Dickinson LSR II flow cytometer.