Monocyte Phenotyping and Intracellular NO Analysis

Monocytes from blood (pre-transmigration and post-transmigration) were stained as described before [8 (link)], with the following modifications. Antibodies were directed against CD33 (Biolegend, San Diego, CA, USA, cat# 366614), CD36 (Biolegend, cat# 336204), CD47 (Biolegend, cat# 323114), CD63 (Biolegend, cat# 353026), CD66b (Biolegend, cat# 392904), CD91 (Invitrogen, cat# 46091942), CD172a (Biolegend, cat# 372106), HLA-DR (Biolegend, cat# 307626), PD-1 (Biolegend, cat# 329952), and PD-L1 (Biolegend, cat# 329738). Viability was determined based on staining with Live/Dead Violet (ThermoFisher Scientific, Wlatham, MA, USA, cat# L34958). Intracellular NO was measured with the cell-permeable probe diaminofluorescein-2-diacetate, as detailed before [5 (link)]. Samples were incubated with antibodies and viability or NO probe in the dark, at 4 °C for 30 min, washed with PBS-EDTA, and centrifuged at 400× g at 4 °C for 10 min, after which the supernatant was removed and samples were fixed overnight at 4 °C in Lyse/Fix buffer (BD Biosciences, San Jose, CA, USA, cat# 558049). Samples were resuspended in 300 µL PBS plus 10 µL counting beads (ThermoFisher Scientific, cat# C36950) to enable absolute cell counting during acquisition on a LSRII flow analyzer (BD Biosciences).