Monocyte HLA-DR Expression Measurement

2.5 × 10⁵ of PBMCs were suspended in 50 μL of phosphate-buffered saline (PBS) and incubated in the dark for 15 min at room temperature with 20 μL of HLA-DR_PerCP, CD11b_PE, and CD14_FITC antibodies (Becton Dickinson, CA, USA). Then, the cells were resuspended in 500 μL of PBS. The monocytes were detected by a three-color flow cytofluorimeter (Beckman Coulter, CA, USA) with positive controls for CD11b_PE and CD14_FITC. Monocyte HLA-DR measurements were expressed as percentages of HLA-DR-positive monocytes and as means of fluorescence intensities (MFI) in relation to the entire monocyte population, thus reflecting the HLA-DR density per cell. Flow cytometry analysis was performed using Kaluza software V1.1 (Beckman Coulter, CA, USA). Setting gates were based on the internal negative population. The figure of analysis strategy for monocyte HLA-DR expression was presented in our previously published paper [13 (link)].