Detecting Intracellular Cytokines in Human PBMC

Flow cytometric detection of intracellular IL-17 and IFNγ in human PBMC was carried out after an extended period of culture as described above. After 13–14 days of culture in the absence or presence of R and corresponding Cit peptides, a staining protocol and a fixation/permeabilization kit (Cytofix/Cytoperm kit with GolgiStop) from BD Biosciences were employed to detect intracellular cytokines as described previously [32 (link)]. In brief, the cells (2 x 10⁶/ml culture medium) were first incubated with 10 ng/ml phorbol-13-myristate acetate (PMA, Sigma-Aldrich), 1 μg/ml ionomycin (Invitrogen, Grand Island, NY, USA), and 1 μl/ml GolgiStop (containing 2 μM monensin) for 4 hours. After blocking Fc receptors with a mouse anti-human CD16/32 mAb, followed by surface staining with a fluorochrome-conjugated mouse mAb against human CD4 (BioLegend, San Diego, CA, USA), the cells were fixed, permeabilized, and stained with fluorochrome-conjugated mouse mAbs to human IL-17A and IFNγ (BioLegend). Flow cytometry was performed using a BD FACS Canto II instrument, and data were analyzed with FACS Diva software (BD Flow Cytometry Systems, San Jose, CA, USA). Non-specific background fluorescence was determined by staining a separate aliquot of human cells with mouse IgG isotype controls (BioLegend) matching the isotypes and fluorochromes of the CD4- and cytokine-specific mAbs.