Pe cy7 anti human cd163

Monocytes were washed in 1× PBS and incubated in blocking solution consisting of FACS buffer (145 mM NaCl, 8.45 mM Na₂HPO₄, 1.83 mM NaH₂PO₄, and 0.1% NaN₃), 5% BSA, and human FcR binding inhibitor (eBioscience, San Diego, CA, USA) for 20 min on ice. After blocking, cells were stained by adding APC anti-human CD14, Alexa Fluor 488 anti-human CD16, PE/Cy7 anti-human CD163, Brilliant Violet 605 anti-human CD86 (BioLegend, San Diego, CA, USA), and APC-R700 anti-human CD80 antibodies (BD Biosciences, San Jose, CA, USA) or isotype controls on ice. Cells were then washed in 1× PBS and analyzed by flow cytometry using an LSRFortessa cell analyzer and BD FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA).
For viability studies, monocytes were washed after incubation with the APC anti-human CD14 antibody as described above. Cells were then stained with phycoerythrin-annexin V (BD Pharmingen, Franklin Lakes, NJ, USA) and either Sytox Blue dead cell stain (Life Technologies, Carlsbad, CA, USA) or propidium iodide (PI) dead cell stain (Thermo Fisher Scientific, Waltham, MA, USA) to detect dead and dying cells. After staining, cells were analyzed by flow cytometry as described above. Double negative cells represent live cells, whereas double positive and single positive cells represent dead and/or dying cells.