Anti human cd68

The cells collected from the peritoneal fluid after 24 h of culture for adherence were fixed with a buffer containing paraformaldehyde (554722, BD Cytofix/Cytoperm USA), washed two times with 1× BD wash buffer (554723, BD Perm/Wash, USA), centrifuged at 350 × g for 5 min, and then the supernatant was discarded. For membrane cytokines, such as CD163 (anti-human CD163; 333606, BioLegend, USA) and CD86 (anti-human CD86; 374216, BioLegend, USA), cells were incubated in diluted primary antibody buffer in the dark for 20 min at about 26°C. For intracellular cytokines, such as CD68 (anti-human CD68; 333806, BioLegend, USA) perm buffer (554723, BD Perm/Wash, USA) was used for 15 min for permeabilization, and the supernatant was discarded after centrifuging at 350 × g for 5 min. Cells were incubated in diluted primary antibody buffer in the dark for 30 min at 4 °C, washed twice with wash buffer, and centrifuged at 350 × g for 5 min. The cells were then resuspended in perm buffer for FCM analysis.

To characterize monocytes, one million PBMCs in 1 mL R10 were cultured with 1 ng/mL IFN-γ or medium alone at 37°C, 5% CO₂ for 24 hours. Cells were then stained with fluorochrome-conjugated anti-human CD14, anti-human CD68, anti-human human leukocyte antigen (HLA)-DR, and anti-human interferon γ receptor 1 (FcγR1, CD119) (all from Biolegend, San Diego, CA, USA) and then counted for monocyte populations and CD14+ cellsby flow cytometry (CyAn ADP analyzer, Beckman Coulter, Fullerton, CA, USA) using Summit software (Fullerton, CA, USA). Gates were set on monocyte population on forward scatter versus side scatter dot plot.