Figure 2
Characterization of purified exosomes. (a) Characterization of equal amounts of purified exosomes by protein marker antibody array. The exosome marker array detects 8 known exosome markers namely – CD63, Immunoglobulin superfamily, member 8 (CD81), Programmed cell death 6 interacting protein (ALIX), Flotillin 1 (FLOT1), Intercellular adhesion molecule 1 (ICAM1), Epithelial cell adhesion molecule (EpCam), Annexin A5 (ANXA5) and Tumor susceptibility gene 101 (TSG101). Cis-Golgi matrix protein marker GM130 serves as control to monitor cellular contamination in exosome preparations. PC stands for positive control. (b) Western blot of equal amounts of CD63-purified exosomes using the exosome markers ALIX and CD63. (c) Absolute size determination and quantification of RSV exosomes either reagent enriched or CD63-purified by NanoSight LM10 analysis. The particles were tracked and sized based on Brownian motion and the diffusion coefficient. The mean and mode diameter of exosomes particles are shown. The absolute count of exosome particles was determined and expressed as particles/ml. (d) Exosomes were enriched from 24 h cell supernatants and equal volumes were analyzed for CD63 expression by Western blot assay. Right panel represents densitometric analysis of three independent experiments. ‘*’ Indicates a statistically significant difference (P value < 0.05) comparing RSV exosomes versus mock.
