Quantification of monocyte HLA-DR was performed according to the methods of Demaret et al. [21 (link)]. Briefly, whole blood was incubated with BD Quantibrite Anti-HLA-DR/Anti-Monocyte Stain (Becton Dickinson, San Jose, CA, USA), lysed using RBC Lysis Buffer (BioLegend, San Diego, CA, USA), and fixed in 2 % paraformaldehyde. Samples were acquired on a FACScan (Becton Dickinson, San Jose, CA, USA) with a five-color upgrade (CyTech, Fremont, CA, USA). Flow files were acquired and analyzed in CellQuest Pro (Becton Dickinson, San Jose, CA, USA). Antibodies bound per cell (ABC) were calculated by standardizing HLA-DR geometric mean fluorescence intensity (GMFI) of monocytes to BD Quantibrite-phycoerythrin (PE) beads (Becton Dickinson, San Jose, CA, USA) (Fig. 1 ).![]()
Gating strategy for determining monocyte HLA-DR levels. A monocyte gate was created by first gating upon forward and side scatter cell properties (upper right panel) and then further refining the monocyte gate by determining positivity for CD14 (lower left panel). Geometric mean fluorescence intensity (GMFI) data were then collected from the monocyte population in the HLA-DR channel (PE) (lower right panel) and compared against a Quantibrite Bead Reference (Becton Dickinson, San Jose, CA, USA) (upper left panel) to yield average per cell HLA-DR intensity. HLA human leukocyte antigen, PE phycoerythrin