Hypoxic enhancement of exosome release by breast cancer cells. (A) MCF7, SKBR3 and MDA-MB 231 cells were cultured at 1% O2 for 48 hours. Exosomes were isolated from conditioned media by ExoquickTM precipitation. Nanoparticle concentrations were determined by NTA and expressed relative to the normoxic control. Data for each sample were derived from four different videos and analyses (n ≥ 3; ± SEM). (B) MCF7, SKBR3 and MDA-MB 231 cells were cultured at 0.1% O2 for 24 hours and exosomes were isolated and analysed as described in (A). (C) CD63 immunoblot of MCF7 ExoquickTM precipitants from a 48 hour culture under normoxia or 1% O2, including band intensity quantitation.(D) CD63 immunoblot of MDA-MB 231 ExoquickTM precipitants from a 24 hour culture under normoxia or 0.1% O2, including band intensity quantitation. (E, F) Nanoparticle size distribution profiles obtained by NTA for hypoxic (0.1% O2) MCF7 ExoquickTM precipitants were normalised to final cell counts for normoxia and hypoxia (E) and relative nanoparticle size distribution profiles were obtained by normalising to total nanoparticle concentration (F). All CD63 immunoblots were performed under non-reducing conditions as described previously . Annotations *, **, and *** correspond with P values <0.05, <0.01 and <0.001 respectively.