Rat anti cd45 percp cy5.5 clone 30 f11

Cells were obtained from harvested tumors as described above, and stained with LIVEDEAD® Fixable Violet and rat anti-CD11b-BV711 (clone M1/70, BioLegend), rat anti-F4/80-AF488 (clone BM8, BioLegend) and rat anti-CD45-PerCP-Cy5.5 (clone 30-F11, eBioscience) antibodies. CD45⁺CD11b⁺ cells were sorted using a 5-laser FACSAria Fusion SORP cell sorter (Becton Dickinson). FSC, SSC and CD45 (695/40) fluorescence were assessed using the 488 nm laser, whereas LD (450/50) and CD11b (710/50) fluorescence were measured with the 405 nm laser. All gates were set based on Fluorescence Minus One (FMO) controls and the temperature was kept at 4°C throughout the entire process.

After blocking and live-dead staining, tumor-derived and cultured immune cells (BMMs, T cells and BMDCs) were stained with the following antibodies (all diluted 1:100) for 30 min at 4°C: rat anti-CD11b-BV711 (clone M1/70, BioLegend), rat anti-CD11b-PE-Cy7 (clone M1/70, eBioscience), rat anti-CD45-V500 (clone 30-F11, BD Biosciences), rat anti-CD45-PerCP-Cy5.5 (clone 30-F11, eBioscience), rat anti-F4/80-APC-Cy7 (clone BM8, BioLegend), rat anti-F4/80-AF647 (clone BM8, BioLegend), rat anti-Ly6G-BV605 (clone 1A8, BioLegend), rat anti-Ly6C-AF700 (clone HK1.4, BioLegend), CD3-APC (clone 17A2, BioLegend), CD8a-APC/Cy7 (clone 53-6.7, BioLegend), CD4-APC/eFluor780 (clone GK1.5, eBioscience), CD40-PE (clone: 3/23, BD Biosciences), CD86-PerCP-Cy5.5 (clone GL-1, BioLegend), CD80-FITC (clone 16-10A1, BioLegend), MHC II-AF647 (clone M5/114.15.2, BioLegend) and CD11c-PacBlue (clone N418, BioLegend). All samples were analyzed with an Attune NxT apparatus (Life Technologies). Compensation was performed using single-stained OneComp eBeads (Thermo Fisher Scientific).