Sc 15363

EVs were collected from 50mL of MIA PaCa-2 cells supernatant cultured for 72h in RPMI medium supplemented with EVs-depleted FBS. EVs isolation was performed as previously described. Then, EVs pellet was resuspended in 100μL of PBS 1X and divided into four conditions for the analysis of CD63, CD81, CD82 and Rab5. A similar protocol to the one performed for the analysis of Evs by flow cytometry was done until detection in ImageStream^× MarkII Imaging Flow Citometry (Luminex Amnis Image Stream Multispectral Imaging Flow Cytometer, RRID:SCR_018589). Primary antibodies used were: anti-CD63 1:400 (Santa Cruz Biotechnology Cat# sc-15363, RRID:AB_648179), anti-CD81 1:400 (Abcam Cat# ab109201, RRID:AB_10866464), anti-CD82 1:400 (Abcam Cat# ab59509, RRID:AB_2076398) and anti-Rab5 1:400 (Cell Signaling Technology Cat# 46449, RRID:AB_2799303). Secondary antibodies used were: Donkey anti-mouse IgG Alexa Fluor 488 1:500 (Molecular Probes Cat# A-21202, RRID:AB_141607) and Goat anti-rabbit IgG Alexa Fluor 488 1:500 (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217). For each sample, control refers to EVs incubated with secondary antibody only.

Cells were lysed in lysis buffer supplemented with PIs, for 15 min at 4 °C, centrifuged at 12000g for 15 min at 4 °C and the supernatant collected and stored at −20 °C until further use. EV (exosomes) and cell lysate protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Scientific MA, USA).
For immunoblotting, total EV, exosomal, and viral proteins or 30 μg of cell lysates were separated by 12% SDS-PAGE and transferred to PVDF membrane (Millipore). Goat polyclonal antibodies against Hsc70 (sc-1059, Santa Cruz Biotechnology, CA, USA), actin (sc-1615), rabbit polyclonal against calnexin (sc-11,397), CD63 (sc-15,363), alpha-tubulin (ab4074, Abcam, UK), VSV-G (ab1874, Abcam), HIV-1 Nef (NIH AIDS Reagent Program, 2949), and HIV-1 gp120 (NBP1–76371, Novus Biologicals, CO, USA); or mouse monoclonal against acetylcholinesterase (AChE, MAB303, Millipore), flotillin, (610820, BD Bioscience, NJ, USA), GFP (sc-9996), p24/Gag (ab9071, Abcam), cytochrome c (556432, BD Biosciences), and Alix (2171S, Cell Signaling, MA, USA) were used as the primary antibodies; and appropriate HRP-conjugated anti-goat, anti-rabbit, or anti-mouse (Jackson Immunoresearch, PA, USA) were used as the secondary antibodies. Membranes were developed by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).