Paraformaldehyde (pfa)

To verify the ability of map‐PD‐L1 to anchor to the cell membrane, we incubated rgEC with 5 μg/ml map‐PD‐L1 or PD‐L1 at 37°C for 1 h. Cells were then fixed in 4% paraformaldehyde (C01‐06002; Bioss), permeabilized with 0.1% Triton X‐100 (C03‐03001; Bioss) in phosphate‐buffered saline (PBS), and incubated with rabbit anti‐PD‐L1 polyclonal antibody (20 μg/ml; NBP1‐76769; Novus Biologicals) overnight at 4°C in humidified chambers. The cells were then incubated with Alexa Fluor‐conjugated secondary antibodies (1:100; CA11012S; Invitrogen) for 1 h at room temperature. Sections were visualized with 4′‐6′‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich) and then examined using confocal laser scanning microscope (Zeiss LSM880).

Cell viability was examined by Cell Counting Kit-8 (CCK-8; Monmouth Junction, NJ, MedChemExpress, USA) and colony formation assay. After 48 h of transfection, transfected cells (2 × 10³ cells/well) were seeded into a 96-well plate and cultured at 37 °C, with 5% of CO₂ for 6 h, 24 h, 48 h, 72 h, 96 h. The 10 µl CCK-8 reagent was added to each well and incubated at 37 °C for 2 h in dark, the absorbance at 450 nm was measured by a microplate reader (Molecular DevicesCMax Plus, USA). For colony formation assay, after 48 h of transfection, transfected cells (4 × 10² cells/well) were seeded into 6-well plates for about 2 weeks and the medium was exchanged every other day. The cell colonies were fixed using 4% Paraformaldehyde (Bioss, Beijing, China) for 15 mins and stained using crystal violet (Sigma Life Science) for 30 mins at room temperature. The colony number was counted by manually.