Intracellular Cytokine Staining of CD4+ T Cells

CD4 T cells were collected and where indicated, stimulated with PMA (50 ng/ml; Sigma) and ionomycin (750 ng/ml; Calbiochem) for 5 hours in the presence of Golgi Plug (brefeldin A, BD biosciences) or with cytokines only for 12 hours, with GolgiPlug added for the last 5 hours. Intracellular staining was performed as previously described [10 (link)]. LIVE/DEAD Fixable Green Dead Cell Stain (Invitrogen) was used extracellularly to exclude dead cells in flow cytometric analyses. Phycoerythrin (PE)-conjugated anti-CD90.1 (OX-7) and anti-IL-17A (TC11-18H10) were purchased from BD Biosciences; allophycocyanin (APC)-conjugated anti- IFNγ (XMG1.2), PE-Cy7-conjugated anti-CD4 (GK1.5), and PE-conjugated anti-IL-18Rα (P3TUNYA) and streptavidin-PE were purchased from eBioscience. Biotin-conjugated anti-mouse IL-1R1 (JAMA-147) was purchased from Biolegend. Samples were acquired on an LSRII instrument (BD Biosciences) and data was analyzed using CellQuest Pro (BD Biosciences) or FlowJo software (Tree Star Inc.).