Tissue dissociator

Single-cell suspensions were washed with PBS and stained with FITC-conjugated PD-L1 at 4°C for 2 hours (5 μL / 2 × 10⁵ cells in 100 μL 0.2% BSA). After washing with PBS, samples were analyzed on BD FACSuite (BD Biosciences). Mouse tumor tissues were first separated into single cells using a tissue dissociator (Miltenyi Biotec). Single cells were stained with antibodies at room temperature for 30 minutes (1 μL / 2 × 10⁵ cells in 100 μL 0.2 % BSA) and then treated as described above. Mouse blood samples were incubated with antibodies at room temperature for 30 minutes (1 μL / 50 μL blood sample). Blood samples were lysed in red blood cell lysis buffer according to standard lysis buffer kit (BD Biosciences) protocol and flow cytometry analysis was performed as described above.

White blood cells were isolated from mouse lungs, spleen, and liver using enzymatic and mechanical dissociation. After slicing the spleen into small fragments of ~2 mm, in PBS, a 10 ml syringe and plunger was used to release the cells further. For lung, tissue chunks were first incubated in 0.5 mg/ml Collagenase D and 20 U/ml DNase (Merck) for 20 min, followed by mechanical dissociation using Miltenyi tissue dissociator. Liver was perfused with 1 mM EDTA in PBS, isolated, and mechanically homogenized by using Miltenyi tissue dissociator. Dissociated tissue samples were then filtered through a 70 μm nylon filter (Miltenyi Biotec). For lungs and liver, the cells were resuspended in 40% Percoll PLUS density gradient medium (GE Healthcare) and overlaid on 70% Percoll Plus medium and centrifuged at 500 g for 30 min at 20°C The interphase containing lymphocytes was collected, washed and subjected to lysis of red blood cells using ACK lysis buffer, together with splenic cells. The isolated cells were then stained as described below.

CD11b⁺ primary microglia were isolated from adult mice aged around 7 months (male and female) and cultured as follows. Mice were euthanized with CO₂ following the Purdue University Animal Care and Use Committee guidelines and brains were transcardially perfused with ice-cold PBS. The perfused brains were dissected and cut into small 1 mm³ pieces before homogenizing them in Dulbecco's Phosphate Buffered Saline⁺⁺ (i.e. with Ca²⁺ and Mg²⁺ ions) containing 0.4% DNase I on a tissue dissociator (Miltenyi Biotec) at 37 °C for 35 min. The cell suspension was filtered through a 70 μm filter and myelin was removed two times, first using Percoll PLUS reagent followed by myelin removal beads using LS columns (Miltenyi Biotec). After myelin removal, CD11b⁺ cells were selected from the single cell suspension using the CD11b beads (Miltenyi Biotec) as per the manufacturer's instructions. The CD11b⁺ cells were finally resuspended in microglia growth media made in DMEM/F12 (Corning Cat. #MT15090CV), further diluted in TIC (TGF-β, IL-34, and cholesterol) media^{40 (link)} with 2% FBS before seeding 0.1 × 10⁶ cells per 500 μL in a well of a 24-well plate (Corning Cat. #353847). The cells were maintained in TIC media at 37 °C and 10% CO₂ with media change every other day until the day (around 10–14 div) of the phagocytosis assay (around 10–14 div).