Pe conjugated anti mouse gr 1

Mice bearing B16F10 and LLC tumors approximately 1000 mm³ in volume were euthanized, and mononuclear cells were isolated from peripheral blood collected via eye bleeding and from the bone marrow obtained by flushing femurs. A single cell suspension of the tumors was prepared by mincing the tumors with a razor blade, homogenizing them by mechanical disruption, and incubating the homogenized tissue in 1 mg/ml of collagenase/dispase (Roche) and DNase I (Sigma) in RPMI-1640 medium as described previously [25] (link). Red blood cells were removed by incubation in ammonium-chloride-potassium (ACK) lysis buffer (ThermoFisher Scientific), and cells were incubated with 1 μg of anti-mouse CD16/32 antibody (eBioscience) per 10⁶ cells to block Fc receptors. Cells were then stained with PE-conjugated anti-mouse Gr-1 (1:200), FITC-conjugated anti-mouse CD11b (1:100), PE-conjugated anti-mouse F4/80 (1:100, eBioscience), and FITC-conjugated anti-mouse CD206 (1:50, BioLegend) antibodies. Before data acquisition on a FACS Canto II cytometer and analysis using FlowJo software (Tree Star), dead cells were excluded from all samples by staining with the Live/Dead Fixable Far Red Dead Cell Stain kit (ThermoFisher Scientific) following the manufacturer’s instructions.