Human colon carcinoma cell line LIM1863 release two populations of exosomes (A33-Exos and EpCAM-Exos) and shed microvesicles (sMVs).

(A) LIM1863 cells were grown in RPMI-1640 medium supplemented with 5% FCS (exosome-depleted), 100 U/ml penicillin and 100 µg/ml streptomycin in CELLine Bioreactor classic flasks and the culture medium (CM) collected. sMVs were first isolated from the CM (yield: ∼20 mg protein, 72 µg RNA). Next, A33-Exos were isolated from the sMV-free CM via anti-A33 antibody capture (yield: ∼3 mg protein, 8.8 µg RNA) and EpCAM-Exos were isolated from the A33-Exos-depleted CM using EpCAM-coupled magnetic beads (yield: ∼3 mg protein, 9.2 µg RNA). (B-D) Electron microscopy images of sMVs (B), A33-Exos (C) and EpCAM-Exos (D) showing a size of 150–300 nm diameter and 40–100 nm diameter for sMVs and A33−/EpCAM-Exos, respectively. Scale bar: 100 nm (n = 3). (E) Western blot of EVs (10 µg protein) for A33, Alix (PDCD6IP), TSG101, and CD9. (F) Total RNA electropherogram analysis (Agilent Bioanalyzer) from LIM1863 cells and derived EVs. Y axis of electropherogram is arbitrary fluorescence unit intensity (FU) and x axis is migration time in seconds (s) and nucleotides (nt).