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).