To validate the changes in class II HLA observed at transcriptomic level with IFN-γ and LPS treatment, we assessed class II expression on primary monocytes by multiparametric flow cytometry. Monocytes were stimulated, as described, before being washed and stained with violet viability dye (Invitrogen) as per the manufacturer’s instructions. To reduce nonspecific staining, a three-step procedure adapted from a protocol shared by R. Apps (National Cancer Institute, National Institutes of Health) was used. Human immunoglobulin G (IgG) was added to each tube, followed by mouse anti-human antibodies directed against HLA-DP (clone B7/21, Abcam), HLA-DQ (clone SPV-L3, Abcam), HLA-DR (clone L243, BD Biosciences), or all class II alleles (clone Tu39 and SK10/SPV-L3 combined) or an isotype control. After 20 min, cells were washed and stained with sheep anti-mouse PE-labeled antibody (Sigma) and sheep IgG.
