Fig. 1

Characterization of EVs from RBCs and uptake of RBCEVs by leukemia cells. a Average concentrations (100,000 × dilution) of RBCEVs from three donors ± SEM (gray) and their size distribution, determined using a Nanosight nanoparticle analyzer. b Representative transmission electron microscopy image of RBCEVs. Scale bar: 100 nm. c Western blot analysis of EV markers ALIX and TSG101; and RBC marker Hemoglobin A (HBA) relative to GAPDH (loading control) in cell lysates and EVs from RBCs. d Western blot analysis of Stomatin (STOM) and Calnexin (CANX) as the markers of RBCEVs and endoplasmic reticulum, respectively, relative to GAPDH, in leukemia MOLM13 cells, NOMO1 cells, RBCs, and RBCEVs. e Western blot analysis of HBA relative to GAPDH in leukemia MOLM13 cells untreated or incubated with 8.25 × 10¹¹ RBCEVs for 24 h. f Representative immunofluorescent images of MOLM13-GFP cells incubated with 12.4 × 10¹¹ PKH26-labeled EVs for 24 h. Scale bar, 20 µm. g FACS analysis of PKH26 in MOLM13 cells that were incubated with 12.4 × 10¹¹ unlabeled or PKH26-labeled EVs with and without Heparin for 24 h. The supernatant of the last wash after PKH26 labeling was used to determine the background. Percentages of PKH26-positive cells are indicated above the gates. h Average percentage of PKH26-positive cells in each condition (mean ± SEM, n = 3 cell passages). P value (***P < 0.001) was determined using Student’s one-tail t-test. In c–e, molecular weights (KDa) of protein markers are shown on the right. Each experiment was repeated two to three times in 2–3 cell passages