Compensation bead particles

To identify specific immune cell subsets in PBMCs following VitD₃ treatment, cells were stained with fluorescently-conjugated monoclonal antibodies; CD4-BUV737, CD45RO-APC, CD161-FITC, CD194-V450, CD196-PE, CD14-BV605, CD19-APC-H7, CD56-BV421, CD282-AF647 (TLR2), CD284-BV786 (TLR4; all from BD Bioscience; San Diego, CA, USA), and anti-TLR7-PE (Gibco Life Technologies, Carlsbad, USA), Zombie Aqua™ Fixable Viability Kit (BioLegend, San Diego, USA). Compensation bead particles were used to account for spectral overlap (BD Bioscience, San Diego, CA, USA) and analyzed using the BD LSRII flow cytometer. Unstained PBMCs and fluorescence minus one (FMO) were used as controls and a minimum of 100,000 events were analyzed per sample gated on live, single cell lymphocyte gate based on FSC and SSC, where the expression of the cell surface molecules was evaluated using FlowJo, LLC v10.4.2 software. Refer to Supplementary Figures 1–4 for gating strategies.

To identify specific immune cell subsets in PBMCs following 1,25(OH)₂D₃ treatment, the cells were stained with fluorescently-conjugated monoclonal antibodies CD4-BUV737, CD14-BV605, and CD3-BV510 (BD Bioscience; San Diego, CA, USA). Compensation bead particles were used to account for the spectral overlap (BD Bioscience, San Diego, CA, USA) above and analyzed using the BD LSRII flow cytometer. Unstained PBMCs were used as a control and a minimum of 20,000 events were analysed per sample gated on live, single cell lymphocyte gate based on FFS and SSC, where the expression of the cell surface molecules were evaluated using MFI values using BD FACSDiva 8.0.1 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).