Phagocytosis Assay for CD11b+ Cells

For isolation of CD11b⁺ cells of Gl261 tumors from ICB R, ICB NR, and C mice, myelin was removed of tumor single-cell suspension with myelin removal beads II (Miltenyi Biotec; 130-096) according to the manufacturer’s instruction. Subsequently, CD11b⁺ cells were purified using MagniSort™ Mouse CD11b Positive Selection Kit (eBioscience; 8802-6860-74). Ex vivo phagocytosis of CD11b⁺ cells was assessed as previously described^{22 (link)}. In brief, CD11b⁺ cells were plated onto ultra-low attachment 96-well plates (Corning) and incubated at 37 °C, 5% CO₂ for 20 min to allow for cell resting. CD11b⁺ cells were subsequently cultured at 37 °C, 5% CO₂ for 2 h with pHrodo™-red Staphylococcusaureus BioParticles (Thermo Fisher) according to the manufacturer’s instruction. Phagocytosis was assessed by flow cytometry analysis for pHrodo-red⁺ cells of macrophages (CD45^highCD11b⁺ cells) and microglia (CD45^lowCD11b⁺ cells). PD-L1 was blocked during incubation with pHrodo™-red S. aureus BioParticles with 20 µg ml⁻¹ anti-PD-L1 (10 F.9G2; BioXCell).