Ionomycin calcium salt from streptomyces conglobatus

Surface markers on cells were stained with Abs in PBS with 2% bovine growth serum (FACS buffer) at 4°C for 20 min. For T_FH staining, cells were stained with purified CXCR5 for 1 h in FACS buffer + 0.5% BSA + 2% normal mouse serum (NMS), followed by biotin-goat anti-rat IgG for 30 min in FACS buffer + 2% NMS, and then streptavidin and other surface-staining Abs for 30 min in FACS buffer + 2% NMS at 4°C. For intracellular cytokine/molecule detection, splenocytes were restimulated ex vivo with 2.5μg/ml gp150_67–83 peptide (LSNNNPTTIMRPPVAQN) or 50ng/ml PMA (Sigma-Aldrich, Milwaukee, WI) and 1μg/ml ionomycin calcium salt from Streptomyces conglobatus (Sigma-Aldrich) in complete medium with 10U/ml rIL-2 and 10μg/ml brefeldin A (Sigma-Aldrich) at 37°C for 5h. Subsequently, cells were stained with Abs against surface markers for 20 min at 4°C, followed by fixation with 1% formaldehyde at 4°C for 20 min, and then stained with Abs against IFN-γ, TNF-α, IL-2 or GzmB in 0.5% saponin solution at 4°C for 30 min. For transcription factor detection, Foxp3/Transcription Factor Staining Buffer Set (eBioscience) was used.