Flow Cytometric Immunophenotyping of PBMCs

Flow cytometry was performed using the following antibodies (ThermoFisher) directed against human antigens: CD45RB (MEM-55), CD25 (BC96), CD3 (OKT3), CD4 (RPA-T4), CD8 (SK1), CD16 (eBiocb16), CD19 (SJ25C1), CD56 (CMSSB), IL-37 (37D12), and Foxp3 (236A/E7). PBMCs treated with or without MCM were washed with FACS buffer (5% BSA (Sigma-Aldrich) in 1x PBS), and then further prepared for surface or intracellular staining, as described previously.^{24 (link)} For surface staining, 1 μg of fluorescently conjugated antibody specific for the surface marker to be visualized was added to 1 × 10⁶ cells resuspended in 100 μl of FACS buffer for 30 min at 4°C, then washed twice with FACS buffer prior to be either analyzed or further processed. For intercellular IL-37 cytokine staining and Foxp3 staining, the Foxp3 Cytoperm/Cytofix staining kit (BD Pharmingen, San Diego, CA) was used.