Macs mouse tumor dissociation kit

Tumors from each mouse were harvested after 10–12 days of treatment as described above. Single cell suspensions were generated from tumors using the MACS mouse tumor dissociation kit (Miltenyi Biotech Cat. 130–096-730) following manufacturer’s instructions. One million cells per tumor were resuspended in PBS and labeled with Cell-ID Cisplatin (Fluidigm, Cat. 201064) to assess for live/dead cells. For antibody labeling, we used the recommended cell surface staining procedure (Fluidigm) followed by the FoxP3/Transcription Staining Buffer Set protocol (eBiosciences™). Cells were labeled with a panel of 28 metal-conjugated antibodies to determine different immune lineages in addition to memory, trafficking, activation, and exhaustion markers (see Supplementary Table 1 for list of antibodies). After washing and centrifugation, cells were fixed using MaxPar Fix and Perm buffer (Fluidigm, Cat. 201067) and labelled for single cell discrimination with Cell-ID Intercalator-Ir (Fluidigm, Cat. 201192A). Samples were resuspended with 10% EQ four-element calibration beads (Fluidigm, Cat. 201078), and filtered through a 40 μm mesh filter prior to acquisition on a Helios™ mass cytometer (Fluidigm), at a rate of 300–500 events/s.

Prostate tissue was subjected to single cell dissociation using the MACS Mouse Tumor Dissociation Kit protocol and gentleMACS Dissociator (Miltenyi). Suspended cells were blocked with rat serum (012–000-120, Jackson ImmunoResearch), stained with FVS570 viability dye (1 ul/ml, 564995, BD Biosciences) in the dark for 15 minutes at room temperature. Samples were washed with PBS and incubated with Myeloid extracellular antibody panel (Supplementary Table S1) or corresponding isotype panels diluted in Brilliant Stain Buffer (566349, BD Biosciences) in the dark for 30 minutes at 4°C. Cells were washed with FACS buffer (1x PBS, 1% BSA, 2mM EDTA), fixed with 1x Fixation Buffer (420801, BioLegend) in the dark for 20 minutes at room temperature, and stored overnight in FACS buffer at 4°C. Samples were incubated in 1x FoxP3 Fix/Perm Solution (421401, BioLegend) in the dark for 20 minutes at room temperature and washed with 1x FoxP3 Perm Buffer (421402, BioLegend). Cells were resuspended with Myeloid intracellular antibody panel (Supplementary Table S1) or corresponding isotype panels diluted in FACS buffer in the dark for 30 minutes at room temperature under gentle agitation. Cell suspensions were washed with FACS buffer and analyzed with a Gallios flow cytometer (Beckman Coulter Life Sciences). Macrophages were defined as FVS570⁻CD45⁺CD11b⁺F4/80⁺CD68⁺ cells.