Exosomal marker antibody sampler kit

Western blots for common exosomal markers were performed as previously described [22 (link)]. Briefly, exosome isolations were lysed in reducing sample buffer (0.25 M Tris-HCl (pH 6.8), 40% glycerol, 8% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol and 0.04% bromophenol blue) or nonreducing sample buffer (without 2-mercaptoethanol) and boiled for 10 min at 95 °C. Proteins were resolved by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), transferred to polyvinylidene fluoride membranes, blocked in 5% nonfat powdered milk in PBS-T (0.5% Tween-20) and probed with antibodies using Exosomal Marker Antibody Sampler Kit (Cell Signaling, Lot #74220). Protein bands were detected using Odyssey infrared Imaging System (LI-COR Biotechnology, USA).

MV and EXO lysates were prepared in RIPA buffer with Halt^TM Protease and Phosphatase inhibitor cocktail, EDTA-free 100× (Thermo Fisher). MV and EXO lysates were loaded in 4–12% NuPAGE Bis–Tris precast gels (Thermo Fisher), proteins were separated and transferred onto a nitrocellulose membrane (Amersham Protran, USA). Membranes were incubated overnight at 4 °C with the following antibodies: anti-P2X7 (1:300, P8232, Sigma-Aldrich), anti-calnexin (1:5000, GTX109669, GeneTex), anti-Alix (3A9) and anti-flotillin-1 (1:1000, Exosomal Marker Antibody Sampler Kit, Cell Signaling Technology). Secondary anti-rabbit (1706515, BIORAD) or anti-mouse (1706516, BIORAD) antibodies were applied at a 1:3000 dilution.

Total protein lysates were prepared from HAE and EVs in freshly prepared lysis buffer (1% Triton, 25 mmol/L Tris pH 7.4, 150 mmol/L NaCl) containing protease inhibitors (complete; mini, EDTA-free; Roche Biochemicals, Mannheim, Germany) for 30 min at 4 °C. Five µg of prepared lysates were solubilized in Laemmli reducing sample buffer (Bio-Rad, Hercules, USA) and separated on a 10% precast Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA, USA). Proteins were electro transferred onto PVDF membrane (Millipore, Burlington, MA, USA), and the membranes were blocked in 5% (w/v) skim milk powder in Tris-buffered saline with 0.05% (v/v) Tween-20 (TBST) for 1 h at RT. Membranes were probed with anti-flotilin-1, anti-CD9, anti-HSP70, and anti-annexin V from the Exosomal Marker Antibody Sampler Kit (Cell Signaling Technology, Danvers, MA, USA) diluted in TBST for 1 h. Blots were rinsed and incubated in horseradish peroxidase-conjugated goat anti-sheep IgG (Bio-Rad, Hercules, MA, USA; 1:10,000) or goat anti-mouse IgG (Bio-Rad, Hercules, MA, USA; 1:10,000) for 1 h at RT with shaking. Antibody staining was visualized by chemiluminescence (ECL Plus Western blotting detection reagents, GE Healthcare, Pittsburgh, PA, USA).