Cd45 percp cy5.5 30 f11

Fc-block was added to dissociated tumor cell suspensions at 1:200 dilution and incubated for 20 min on ice. Three panels of flow cytometry antibodies were used for leukocyte staining—Panel 1: CD11b-ef450 (M1/70) #48-0112-82 Invitrogen, MHCII-FITC (M5/114.15.2) #11-5321-81, F4/80-AF700 (BM8) #56-4801-82, CD11c-PECy7 (N418) #25-0114-82 eBioscience, CD45-APC (30-F11) #10311 and Live/Dead-AmCyan (#423101) Biolegend; Panel 2: CD45-PerCP-Cy5.5 (30-F11) #103132, CD3e-APC (145-2C11) #100312, B220 BV605 (RA3-6B2) #103243, PD-1-PE (29F.1A12) #135205, CD4-BV421 (GK1.5) #100437, CD8a-APC Fire 750 (53-6.7) #100766 and Live/Dead-AmCyan (#423101) Biolegend; Panel 3: CD206-FITC (MR5D3) #MA5-16870, CD11b-ef450 (M1/70) #48-0112-82 Invitrogen, F4/80-AF700 (BM8) #56-4801-82 eBioscience, intracellular Arg1-APC (#IC5868A) R&D systems, MHCII-APC-Cy7 (M5/114.15.2) #107652, CD45-PerCP-Cy5.5 (30-F11) #103132 and Live/Dead-AmCyan (#423101) Biolegend.

Lungs were digested with 0.52 U/ml Liberase TM (Roche) and 60 U/ml DNase I (Roche) in RPMI 1640 (Cellgro), at 37°C for 30 min. Lung leukocyte enrichment was performed by using a 30% Percoll gradient and the cell suspension obtained after erythrocyte lysis. Single cells were then incubated with anti-mouse CD16/32 (93, TruStain fcX, BioLegend) to block Fc receptors. Staining was performed with Fixable Viability Dye eF780 (eBioscience), to identify dead cells, and the following fluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs): CD45-PerCP/Cy5.5 (30-F11), CD4-BV785 (GK1.5) (all from BioLegend), and CD3e-BUV395 (145-2C11) (from BD Biosciences). At least 1,000,000 events were recorded for each sample. Only viable and non-doublet cells were considered. Flow cytometric analysis was performed using a LSRFortessa X-20 flow cytometer (BD Biosciences) and FlowJo software (Tree Star).