2.7 Cell Culture for Immunostaining and Flow Cytometric Analyses

Purified αPoIL-17A or αPoIFNγ mAbs were labeled with AF647 according to the manufacturer’s instructions using the Alexa Fluor^® 647 Protein Labeling Kit (ThermoFisher Scientific, Waltham, MA). For cell culture, frozen PBMCs were thawed and cultured overnight in blastogenic medium in 6 well plates at 4 x 10⁶ cells/well. Cells were stimulated for 5 hrs with BD Leukocyte Activation Cocktail (BD Biosciences, San Diego, CA), which is a ready-to-use polyclonal cell activation mixture containing PMA/Iono, and a protein transport inhibitor (Brefeldin A). For cell surface staining, Fc receptors were first blocked for 30 min at 4°C in complete Flow Cytometry Medium (FCM) [PBS-BSA with 20 mM of Sodium Azide, and 1% normal Rabbit Serum]. For dead/viable cell exclusion, the cells were stained with the fixable viability stain 520 (FVS) (BD Biosciences, San Diego, CA) in PBS for 7 min at 37°C and washed twice with PBS-BSA. Alternatively, the cells were stained with the VivaFix cell viability dye (Bio-Rad, Hercules, CA) in PBS for 30 min at room temperature, then washed twice with PBS-BSA.

For intracellular staining, after staining for dead/viable cell exclusion as noted above, Fc receptors were blocked for 30 min at 4°C in complete FCM. The cells were then stained with PE-conjugated αPoCD3 mAb (BD Biosciences, San Diego, CA) for 30 min at 4°C in normal FCM, fixed for 30 min at 4°C in Fixation & Permeabilization Buffer (BD Biosciences, San Diego, CA), washed twice with 1x Permeabilization Buffer (BD Biosciences, San Diego, CA), and labeled with AF647-conjugated αPoIL-17A or αPoIFNγ mAb ( Table 1 ) for 30 min at 4°C in 1x Permeabilization Buffer. The cells were washed twice with 1x Permeabilization Buffer and re-suspended in normal FCM. For flow cytometric analyses, data on labeled cells were acquired either on an Accuri C6 or an Accuri C6 Plus flow cytometer (BD Biosciences, San Diego, CA) and analyzed using FlowJo Software, version 10.7.1 (BD Biosciences, San Jose, CA), gating on live lymphocytes and live T cells.