Anti sirpα

Harvested cells were stained with antibodies including: anti-Ly6G (1:200 dilution; BioLegend, no. 127606, 127610, or 127618), anti-CD11b (1:200 dilution; BioLegend, no. 101206), anti-CD11a (1:200 dilution; BioLegend, no. 153103), anti-SIRPα (1:200 dilution; BioLegend, no. 37210), anti-CD62L (1:200 dilution; BioLegend, no. 104428), anti-PD-L1 (1:200 dilution; BioLegend, no. 124312), anti-CXCR2 (1:300 dilution; BioLegend, no. 149604), anti-ICAM1 (1:300 dilution; BioLegend, no. 116121), anti-CD29 antibodies (1:300 dilution; BioLegend, no. 102221). In some analysis mentioned in the results, cells were treated with a fixation kit (BD Biosciences), subsequently stained with anti-STAT1 (1:100 dilution; Cell Signaling, no. 80916S), anti-p-STAT1 (Tyr701) (1:100 dilution; Cell Signaling, no. 8009S), anti-p-Src (Tyr416) (1:20 dilution; ThermoFisher, no. MA5-28055), or anti-Fyn (1:50 dilution; Santa Cruz, no. sc-434 FITC). Surface phenotype, transcription factor and intracellular protein levels of Ly6G⁺ neutrophils were analyzed using FACSCanto II (BD Biosciences). Neutrophil viability was assessed by staining and flow analysis with the annexin V/PI kit (1:4,000 dilution; Thermo Fisher Scientific, no. P3566) as described previously^{74 (link)}.

BM cells isolated from WT C57BL/6 mice and Tram^–/– mice (CD45.2⁺) were cultured with complete RPMI 1640 medium supplemented with M-CSF (10 ng/mL) for 3 days. The cells were harvested, stained with anti-Ly6G (1:200 dilution, BioLegend, no. 127606) and anti-CD11b (1:200 dilution, BioLegend, no. 101226) antibodies, and labeled with PI. CD11b⁺Ly6G^–PI^– cells were purified with SH800 cell sorter (Sony) to obtain donor BMMs. BM cells isolated from B6 SJL mice (CD45.1⁺) were cultured in 12-well plates (5 × 10⁵ cells/well) with complete RPMI 1640 medium supplemented with M-CSF (10 ng/mL) to serve as recipient cells. After 3 days, floating cells were removed, and Transwell inserts (Corning) were placed in some recipient cell cultures. WT or Tram^–/– donor BMMs were added to the upper chambers of the Transwell inserts (5 × 10⁵ cells/well) or directly added to the recipient BMMs without Transwell inserts (5 × 10⁵ cells/well). After 2 days, the cells were harvested and stained with anti-CD45.1 (1:200 dilution, BioLegend, no. 110730), anti-CCR2 (1:200 dilution, BioLegend, no. 150628), anti–SIRP-α (1:200 dilution, BioLegend, no. 144014), and anti-CD200R (1:200 dilution, BioLegend, no. 123916) antibodies. The surface phenotype of CD45.1⁺ recipient BMMs was determined with flow cytometry.

Exactly 0.5 to 1 × 10⁶ tumour single‐cell suspension or the treated cells were incubated with an antibody cocktail. Following reagents and antibodies were used for flow cytometry analysis, including Zombie Red™ Fixable Viability Kit, anti‐CD45, anti‐F4/80, anti‐CD11b, anti‐CD206, anti‐CD80, anti‐Thy1.2, anti‐CD4, anti‐CD8a, anti‐PD‐1, anti‐Ly6G, anti‐Ly6C and anti‐SIRPα (BioLegend). For intracellular staining, the cells were first stained for membrane‐bound protein, then treated with Fixation/Permeabilisation buffer (BD Pharmingen San Jose, California) and incubated with anti‐TNF‐α, anti‐IL‐6 and anti‐IL‐17A. Flow cytometry was performed on BD FACSAria™ III instruments (BD Biosciences, Franklin Lakes, New Jersey). During running on flow cytometry instrument, 20,000 events were recorded for each sample. Fluorescence compensation was made by single positively stained control on BD compensation beads. Data were analysed by FlowJo software, version 10.4 (Tree Star Inc.). The stained samples were carefully gated according to FMO controls (Fluorescence minus one control). Gating strategies were described in Figure S1.