Attune nxt acoustic focusing flow cytometry platform

Freshly harvested tumors from Trem1^+/+ or Trem1^–/– mice were processed into a single-cell suspension using gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech) in combination with the tumor dissociation kit (Miltenyi Biotech). Cells were stained with fluorochrome–conjugated antibodies according to the manufacturer’s instructions. For surface staining, cells were prepared and suspended in PBS and incubated with following antibodies (all from BioLegend) at 4°C for 45 minutes in dark: TruStain FcX (clone: 93, 101319, 1:50 dilution), anti-F4/80-APC (clone: BM8, 123116, 1:100 dilution), anti-F4/80-FITC (clone: BM8, 123108, 1:100 dilution), anti-CD11b-APC (clone: M1/70, 101212, 1:100 dilution), anti-CD11b-PE (clone: M1/70, 101208, 1:200 dilution), anti-CD11b-APC/Cy7 (clone: M1/70, 101226, 1:100 dilution), anti-Gr1-APC/Cy7 (clone: RB6-8C5, 108423, 1:100 dilution), anti-Ly6C-PE (clone HK1.4, 128007, 1:200 dilution), anti-Ly6C-APC/Cy7 (clone: HK1.4, 128026, 1:100 dilution), anti-Ly6G-PE (clone: 1A8, 127608, 1:200 dilution), and anti-Ly6G-APC/Cy7 (clone: 1A8, 127624, 1:100 dilution). Cells were acquired on the Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and data were analyzed on FlowJo v10.0.

Tumor-infiltrating MDSCs were isolated from freshly harvested B16F10 tumors of Trem1^+/+ or Trem1^–/– mice using the MDSC isolation kit (Miltenyi Biotec) per the manufacturer’s instructions. The purity of MDSCs was over 80%–90% as verified by flow cytometry. CD3⁺ T cells were harvested from spleens of Trem1^+/+ mice and enriched by negative selection via Pan T cell isolation kit (Miltenyi Biotec) per the manufacturer’s instructions. The purified CD3⁺ T cells were stained with CFSE (Thermo Fisher Scientific, C34570, 2 μM) and maintained in complete medium consisting of RPMI (STEMCELL Technologies), 10% heat-inactivated FBS (Hyclone),100 U/mL penicillin, and 100 μg/mL streptomycin (Corning). For proliferation, CD3⁺ T cells were primed using anti-CD3/CD28 beads (Gibco, 11-452-D) for 72 hours according to manufacturer’s recommendation and cocultured with purified MDSCs at a 2:1 ratio. T cell proliferation was subsequently measured by acquiring the extent of CFSE dilution in T cells on an Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and analyzed using FlowJo v10.0.

MDSCs were isolated from Trem1^+/+ or Trem1^–/– tumor-bearing mice as described earlier. ROS were detected using the ROS assay Kit (Invitrogen) per the manufacturer’s instructions. For induced activation experiments, MDSCs were cocultured with 30 ng/mL PMA (Thermo Fisher Scientific, 50-058-20001). ROS formation was acquired on an Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and analyzed using FlowJo v10.0.