Exosome elisa kit

Transfection reagents Lipofectamine and FuGENE6 were purchased from ThermoFisher Scientific (Waltham, MA) and Promega (Madison, WI) respectively. ExoQuick-TC, Exosome ELISA kit, XPACK-RFP and Dot blot antibody array (antibodies for exosome markers of CD63, CD81, ALIX, Flot1, ICMA1, EpCam, ANXAS and TSG101, as well as a cytosolic protein control GM130) were purchased from System Biosciences (SBI, Palo Alto, CA). Human embryonic kidney cells (HEK293) were purchased from Alstem (Richmond, CA) and human glioblastoma cells (U87) were from the American Type Culture Collection (ATCC, Manassa, VA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and puromycin reagent were purchased from ThermoFisher (Fremont, CA). UltraCULTRE medium was purchased from Lonza (Allgendale, NJ).

BMSC from passage 3 were expanded for 5 days to achieve passage 4 and then re-plated in 6 well tissue culture plates in serum free medium at a concentration of 1 million per ml. Exosome secretion inhibitor GW4869 was added at a concentration of 10μM. BMSC were incubated overnight with GW4869 (Santa Cruz Biotechnology), washed and then tested for the inhibition of exosome secretion using the exosome ELISA kit from Systems Biosciences. Briefly, supernatants were collected from BMSC cultures that were either treated with DMSO or GW4869. Exosome enrichment and protein isolation was carried out as per the manufacturer's instructions. The enriched protein was then analyzed for CD81 expression by ELISA. BMSC that were either treated with DMSO or GW4869 were then utilized for transwell assays. Human Th1 cells (0.5 million per ml) were placed at the bottom of the transwell and the upper chambers consisted of BMSC or BMSC pretreated with GW4869 (0.5 million per ml). ATP was added to the culture and the supernatant was collected after 4 hrs. Adenosine production was measured in the supernatant by GC-MS/MS.