Extracellular Vesicle Hemolysis Assay

Hemolysis assay was performed by adopting the protocol as described earlier (25 (link)). Briefly, different concentrations of EVs or other components were mixed with an equal volume of 2% RBC and incubated at 37°C for 60 min. After removing undissolved RBCs by centrifugation (1,500 rpm, 10 min), 100 µL of supernatant was removed to a 96-well plate. Absorbance of the released hemoglobin in the supernatants was determined at 450 nm using a microplate spectrophotometer (Thermo Fisher Scientific). PBS and Triton X-100 (0.1%) were used as negative and positive controls, respectively. Hemolysis rate (%) = (EV group OD450 − negative control OD450) / (positive control OD450 − negative control OD450) × 100%.