Ly6g percp cy5.5 clone 1a8

Cells were washed with FACS medium (5% FCS in RPMI) and non-specific binding sites were blocked by incubating 20 minutes at 4°C with an Fc-blocking antibody (anti-CD16/32, clone 2.4G2). Next, cell suspensions were stained with fluorescent conjugated antibodies for 30 minutes at 4°C. Fluorescent antibodies: CD11b PE-Cy7 clone M1/70, F4/80 FITC clone C1:A3-A, Ly6C APC clone AL-21, Ly6G PerCP-Cy5.5 clone 1A8, CD45 APC-Cy7 clone 30-F11, CD4 BV421 clone GK1.5, CD8 BV510 clone 53–67, NK11 PE clone PK136 (BD Biosciences), CD64 Pe clone X54-5/7.1. (BioLegend), CCR2 Pe clone 475301, MerTK Pe clone 108928 (R&D systems), Ly6B clone 7/4 (AbD Serotec)., TCRb APC clone H57-597, CD49b Pecy7 clone DX5, NKp46 PE clone 29A1.4 (eBioscience). Following washing with FACS buffer they were analyzed on a FACS Canto II flow cytometer (BD Biosciences) and data was processed using FlowJo software (Tree Star Inc.).

After overnight co-culturing (see above) cells were subjected to flow-cytometrical analysis. Briefly, the cells were washed with FACS medium (5% FCS in RPMI) and non-specific binding sites were blocked by incubating 20 minutes at 4°C with an Fc-blocking antibody (anti-CD16/32, clone 2.4G2). Next, cell suspensions were stained with fluorescent conjugated antibodies for 30 minutes at 4°C. Fluorescent antibodies: CD11b PE-Cy7 clone M1/70, F4/80 FITC clone C1:A3-A, Ly6C APC clone AL-21, Ly6G PerCP-Cy5.5 clone 1A8, CD45 APC-Cy7 clone 30-F11 (BD Biosciences), CD64 PE clone X54–5/7.1. (BioLegend), CCR2 PE clone 475301, MerTK PE clone 108928 (R&D systems), Ly6B clone 7/4 (AbD Serotec). Following washing with FACS buffer they were analyzed on a FACS Canto II flow cytometer (BD Biosciences) and data was processed using FlowJo software (Tree Star Inc.).

Cells were resuspended in PBS and stained on ice for 30 minutes in the dark with a fixable viability stain (BD Bioscience). Then, cells were resuspended into the stain buffer (BSA) (BD bioscience) and stained on ice for 30 minutes with various combinations of directly fluorochrome-conjugated antibodies. Antibodies used for flow cytometry were from BD Biosciences, Biolegend, or ThermoFisher, and include CD45 APC-Cy7 (clone 30-F11), CD8a BUV737 (clone 53-6.7), TCRb PE (clone H57-597), CD4 BUV395 (clone RM4-5), CD25 BV711 (clone PC61), CD11b AF700 (clone M1/70), Ly6G PercpCy5.5 (clone 1A8), CD11c PeCy7 (clone N418), Nkp46 FITC (clone 29A1.4), F4/80 PE-CF594 (clone T45-2342) Ly6C BV421 (clone AL-21), CXCR3 APC (clone CXCR3-173), Class I MHC FITC (clone 34-1-25) and PD-L1 BV421 (clone MIH5). For all samples, acquisition was performed on Fortessa flow cytometer (BD). Data were analyzed using FlowJo software (Tree Star).