Live dead fixable aqua violet

Cells were stained 30 min at 4C in a final volume of 100μL staining buffer with antibodies against CD4 (OKT4, Thermo Fisher Scientific), IL-6 (MQ2-13A5, BioLegend), LAG-3 (3DS223H, Thermo Fisher Scientific), CD49b (P1H5, Thermo Fisher Scientific) with DAPI or LIVE/DEAD Fixable Aqua/Violet (Invitrogen) to exclude dead cells. For intracellular staining, cells were treated with the spiked addition of Phorbol 12-myristate 13-acetate (PMA) (50ng/mL, Sigma-Aldrich), ionomycin (1μg/mL, Sigma-Aldrich), GolgiStop (1X, BD Biosciences) and Brefeldin A (1X, BD Biosciences) to media then cultured 5h, subsequently washed in PBS, surface stained, treated with Cytofix/Cytoperm (eBioscience) and subsequent Perm/Wash buffer (eBioscience) steps before incubation with intracellular antibodies for 30 min at 4C. Samples were washed and acquired on a LSRFortessa (BD Biosciences) or the Attune NxT Flow Cytometer (Invitrogen) within 24h and analyzed using FlowJo/v9.9.6. Cell proliferation analysis was carried out by CFSE dilution in polyclonally activated CD4⁺CD25⁻ T cells with carrier or VitD after 72h.
Cell proliferation and activation markers were assessed in purified CD4⁺ cells with CellTrace Violet (CTV, C34556, Invitrogen) and surface staining against CD62L-FITC (MEL-14, BioLegend), CD69-PE-Cy7 (FN50, Invitrogen) or CD25-PE-Cy7 (BC96, Invitrogen) and LIVE/DEAD fixable near-IR (Invitrogen).