Mahas4510 plate

Enzyme‐linked immunospot assay was performed using a ELISpot kit (Mabtech) according to the manufacturer's instructions as described previously.²⁹ Peptide‐stimulated human PBMCs (2 × 10⁵) or mouse lymphocytes (5 × 10³) were cultured in the presence of peptide (3 μg/ml) in a MAHAS4510 plate (Millipore) for 24 h, and the number of IFN‐γ‐producing cells in the culture was measured using an ELISpot kit (Mabtech) according to the manufacturer's instructions. BCIP/NBT plus substrate (Mabtech) was used for detection. Plates were scanned using an automated ELISpot plate reader (Autoimmun Diagnostika GmbH). Spots were counted and analyzed using AID ELISPOT plate reader software (Autoimmun Diagnostika GmbH). For mouse lymphocytes, splenocytes (3 × 10⁵) were used as APC.

For purification of CD11b⁺Ly6C⁺ monocytes/macrophages from tumor tissues, single-cell suspensions from tumor tissues were prepared after 24 h of intratumoral treatment with control, anti-CD47 mAb, cGAMP, or a combination. CD45⁺ cells were isolated from the single-cell suspensions using the MACS isolation system (Miltenyi Biotech) according to the manufacturer’s protocol, and then CD45⁺CD11b⁺Ly6C⁺Ly6G⁻ monocytes/macrophages were sorted using FACSAria II (BD Biosciences). The sorted monocytes/macrophages were cocultured with OT-I cells in a MAHAS4510 plate (Millipore) for 24 h. IFN-γ–producing T cell numbers were visualized by anti-mouse IFN-γ enzyme-linked immunospot assay (ELISpot, catalog no. 3321-2A; Mabtech) according to the manufacturer’s protocol. BCIP/NBT plus substrate (Mabtech) was used for detection. Plates were scanned with an automated ELISpot plate reader (Autoimmun Diagnostika). Spots were counted and analyzed using the AID ELISpot plate reader software (Autoimmun Diagnostika).