Mice were intravenously injected with DiD-labeled nano-Ag@erythrosomes (RBC:B16F10 membrane protein, 20:1; B16F10 membrane protein, 10 μg). After 12 hours, mice were sacrificed and spleen was collected into single-cell suspensions. Splenocytes (2 × 106) were stained with fluorescein isothiocyanate (FITC)–CD11c, phycoerythrin (PE)–CD80, PE-CD86, PE-CD40, PE–MHC II and PE–PD-L1 for DC analysis and FITC-F4/80, peridinin chlorophyll protein (PerCP)–CD11b, PE-CD80, PE-CD86, PE-CD40, PE–MHC II, and PE–PD-L1 for macrophage analysis. The cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences).

The mice were sacrificed at day 12, and tumors were collected and cut into pieces. The tumor tissues were homogenized in PBS containing 1% fetal bovine serum and filtrated through nylon gauze to obtain single-cell suspensions. The primary antibodies used for flow cytometry were purchased from BioLegend. The stained cells were analyzed with the FlowJo software package (version 10.0.7; TreeStar, USA, 2014).

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