Anti cd81 pe

All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Anti-mAbs used for Western blotting against extracellular signal-regulated kinase (ERK)1/2, p-ERK1/2, p38, p-p38 (Thr180/Tyr182), c-Jun N-terminal kinase (JNK), p-JNK (Thr183/Tyr185), IκB-α, p-IκB-α (Ser32), p-Ikkα/β (Ser176/180), and p-Akt (Ser473) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-Gα16 mAb was from Abcam (Cambridge, UK). Anti-F(ab′)2 fragment goat anti-mouse (GAM) IgG (H + L) was from Jackson ImmunoResearch (West Grove, PA, USA). Anti-CD11b-PE, anti-CD62L-PE, anti-CD81-PE, anti-CD9-APC, and anti-CD4-FITC for flow cytometry and anti-phosphotyrosine, anti-β-actin mAb for Western blotting were purchased from BD Biosciences. The mAbs used for cell stimulation were 2A1 (EMR2-specific mAb) (AbD Serotec) and mouse monoclonal IgG1 (Clone 11711) (R&D System) as described previously (18 (link)).

EVs were purified from human plasma by differential centrifugation as reported above. The EVs were resuspended in PBS + 0.1% BSA and then stained with an anti-CD81 PE (clone BD Pharmingen, clone JS-81) or anti-alpha-sarcoglycan (clone AD1/20A6 Monosan) and labelled by goat anti-mouse (GaM) FITC. The cytometric analyses were performed by gating events smaller than 1 mm. Size beads (Ø 1–2 mm Polysciences Invitrogen, and Ø 5.2 mm DakoCytoCount beads) were used to establish the proper gate for events smaller than 1 mm, which include EVs, and to obtain single platform absolute counts [36 (link)].