Quantifying Exosome Markers by Western Blot

For western blot (WB) analysis, equal amounts of EV pellet (30-50 ug, measured by Pierce BCA protein assay kit, CAT 23227) obtained from similar PPP volumes (250 ul) were combined with a 2xlysis buffer (RayBiotech) supplemented with 1% proteinase inhibitor and 1% phosphatase inhibitors (Sigma) containing β-mercaptoethanol (1:20, Biorad). Samples were loaded and separated on 4%–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) and then transferred to Trans-Blot Turbo Mini 0.2 μm Nitrocellulose Transfer Packs (Bio-Rad). The membranes were stained with Ponceau S solution (P7170, sigma) to ensure that proteins transferred from the gel to the membrane (Supplementary Figure S3), were washed and immunoblotted with the appropriate antibodies against exosome markers. Mouse monoclonal anti human-CD63 (ab59479) and CD81 (ab79559) were both used in 1:1000 dilutions. Anti-rabbit, anti-human placental lactogen hormone (hPL) (ab137099; 1;25,000 dilution) and Calnexin (ab10286, 1;10,000 dilution) endoplasmic reticulum (ER) protein, not expected to be enriched in EVs, serve as negative control (all from Abcam, USA). Secondary antibodies (anti-mouse 115-035-146 and anti-rabbit 111-035-144, both in 1:5000 dilution) were purchased from Jackson ImmunoResearch (PA, USA). The blot was imaged and quantified by myECL™ Imager and analyzed by My Image Analysis Software (both from Thermo Fisher Scientific, Waltham, MA USA).