The whole blood was first collected from the orbital sinus of C57 mice and stored in phosphate-buffered saline (PBS) containing EDTA. Then, RBCs were separated from whole blood by centrifugation (2000 rpm, 5 min). The RBC membrane was obtained by a previously reported hypotonic treatment (42). Briefly, deionized water containing EDTA was added into the obtained RBCs, and the mixture was shaken gently for 5 min. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and further centrifuged at 14,800 rpm for 20 min at 4°C. Deionized water containing EDTA was added to the precipitation at the first step, and the solution was sonicated for 10 s, followed by centrifugation at 14,800 rpm for 20 min at 4°C. The procedure was repeated twice. The obtained RBC membrane was washed twice with deionized water containing EDTA to remove the hemoglobin.

To obtain the B16F10 membrane, the cells were suspended with homogenization medium [0.25 M sucrose, 1 mM EDTA, 20 mM Hepes-NaOH, and protease inhibitor cocktail (pH 7.4)]. The cells were sonicated using Selecta Sonopuls for 30 rounds on ice (Ton = 3 s, Toff = 7 s). The solution was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and further centrifuged at 14,800 rpm for 20 min. The obtained B16F10 cell membrane was washed twice with PBS.

To obtain cell membrane from tumor tissue, B16F10 or 4T1 tumor tissues were collected and cut into pieces, followed by sonication using Selecta Sonopuls for 30 rounds on ice (Ton = 3 s, Toff = 7 s). For B16F10 tumor, the mixture was centrifuged at 6000 rpm for 10 min to remove melanin (for 4T1-Luc tumor, this step was skipped), and the supernatant was collected and further centrifuged at 14,800 rpm for 20 min.

The protein amount in the RBC and B16F10 membranes was determined using a BCA kit. The mixture of both membranes was sonicated for 15 min until the mixture solution became transparent. During this step, ice was added into the sonicator to avoid protein denaturation caused by heat. Then, the mixture was gently shaken using a dry bath incubator at 37°C for 30 min before extrusion through a 400-nm membrane.

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