Flow cytometry analysis

Cellular uptake of sEVs and assessment of cell surface and internal antigens were analysed by flow cytometry. To investigate sEV uptake by tumour cells, PKH26-labelled sEVs were incubated with tumour cells for 4 h, after which cells were washed three times with cold PBS and fixed in 4% paraformaldehyde. To assess cell surface and internal antigens, tumour tissues obtained at Day15 were isolated after transcardial perfusion from each treatment group were collected and digested at 37 °C for 60 min in 10 mmol L⁻¹ HEPES buffer with 300 U mL⁻¹ collagenase D, dispase, and 15 U mL⁻¹ DNase I to obtain cell suspensions. After dissociation, the cells were filtered through a 70 μm nylon cell strainer and collected. For flow cytometry, cells were fixed and permeated to allow the entry of fluorescence probes. To avoid nonspecific binding to the Fc receptor, cells were first blocked with an anti-CD16/CD32 antibody (Bio X Cell, catalogue no. BE0307, dilution of 1:200) for 15 min. Then, cells were incubated with various labelled antibodies (anti-IFN-γ-PE at a 1:200; anti-MHC-I-APC at 1:300; anti-CD8a-PE at 1:200; anti-CD86-PE at 1:300; anti-CD45-PerCP at 1:200; anti-CD3-APC at 1:300; or anti-F4/80-APC at 1:300 dilution) according to the manufacturer’s instructions. Cell fluorescence intensity was analysed with a flow cytometer (Gallios 561, Beckman). At least 10,000 events were collected per cell sample. Representative gating strategies for all flow cytometry data are shown in Supplementary Fig. ³⁹.