Flow cytometry
Whole blood samples of 50 μL were stained with fluorescein isothiocyanate (FITC)-labeled antihuman CD14 and either phycoerythrin (PE)-labeled anti-HLA-DR or anti-PD-L1 antibodies (BD Biosciences, La Jolla, CA). After 25 mins of staining at 4°C, erythrocyte lysis was achieved with ammonium chloride, potassium bicarbonate, and EDTA (Sigma Chemical Co., St. Louis, MO) for 6 min. Cells were then washed with Dulbecco Phosphate Buffered Saline (DPBS) (Sigma Chemical Co., St. Louis, MO) and fixed in 300 μL of 1% paraformaldehyde.
For flow cytometric analysis, samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA) within 48 hours of venipuncture. A minimum of 20,000 events were acquired and mean fluorescence intensity (MFI) analyzed using Cell Quest software (Becton Dickinson, San Diego, CA). Appropriate isotype controls were used for each antibody to check for non-specific binding (BD Biosciences, La Jolla, CA).