Multiplex assay

MC38 tumors without IR and three days post-IR were excised from WT and Sirpα^−/− mice. After being weighed, tumors were minced followed by culturing in RPMI-1640 with 10% FBS. After 24 h, the culture supernatants (tumor-conditioned medium) were collected and tested for the presence of MCP-1 and KC chemokines by a multiplex assay (BioLegend), and the capability of driving monocyte/MDSC or PMN chemotaxis by in vitro chemotaxis assays^{49 (link),51 (link)}. Ly6C⁺ monocytes/MDSCs and Ly6G⁺ PMNs (1 × 10⁶ each) isolated from WT mouse bone marrow^{52 (link)} were labeled with CFSE and placed into the upper chamber of the transwell device in a 24-well plate. The collected tumor-conditioned medium (0.5 ml) was added into the lower chamber followed by incubation at 37 °C for 2 h. Chemotaxis of monocytes/MDSC and Ly6G⁺ PMN into the lower chamber was quantified by a SpectraMax iD5 Multi-Mode Microplate Reader with SoftMax Pro 7 Software (Molecular Devices) and calculated against the total cells loaded. Control experiments were performed using medium without culturing tumor tissues.

Prior to (0 h) and after (24 and 72 h) tumor IR, the tumor-bearing WT, and Sirpα^−/− mice were exsanguinated to obtain serum. Cytokines including TNFα, IL-1β, IL-12, and IFNγ in mouse sera were detected by a multiplex assay (BioLegend) according to the manufacturer’s instructions.