Anti cd63 apc

Human serum albumin (HSA), methylglyoxal (MG), D-glucose, Histopaque, sodium citrate, 3,3′,5,5′-tetramethylbenzidine (TMB), phorbol 12-myristate 13-acetate (PMA), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), celestine blue B (CB), 4-chloro-1-naphtol (4-CN), Coomassie G-250, o-dianisidine, hydrogen peroxide (H₂O₂), horseradish peroxidase (HRP), luminol (Lum), lucigenin (Luc), Triton X-100 and all salts and solvents for the preparation of solutions were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acrylamide, methylene bis-acrylamide, tetramethylethylenediamine, ammonium persulphate, Tris and glycine were purchased from Panreac-AppliChem (Darmstad, Germany). Krebs-Ringer buffer solution was purchased from Merck (Kenilworth, NJ, USA). Anti-CD11b-PE, anti-CD63-APC, anti-CD45-FITC antibodies, non-fat dry milk (blotting grade blocker) and HRP-labeled anti-mouse IgG, nitrocellulose membrane were purchased from Bio-Rad (Hercules, CA, USA). Dextran T70 was purchased from Roth (Karlsruhe, Germany). May Grünwald’s Eosin–Methylene Blue solution and Romanowski Azur Eozin stain were purchased from ECOlab (Moscow, Russia).

Isolation of extracellular vesicles (EVs)was performed by differential ultracentrifugation, as described previously [25 (link)]. The pellet obtained by ultracentrifugation at 100,000 g for 70 min corresponding to the fraction containing the EVs was resuspended in 0.1 μm filtered PBS with sucrose at 1% (Sigma-Aldrich). The presence of EVs in the sample was detected by Transmission Electron Microscopy (TEM) JEM-1011 (JEOL, Japan) at 100 kV with a previous fixing with 2% paraformaldehyde and dyed with 2% phosphotungstic acid. The EVs and Taxol-encapsulated EVs were characterized by a Pierce™ bicinchoninic acid assay kit (Thermo Fisher Scientific, USA) for total protein quantification. The size distribution and particle concentration were determined by Nanoparticle Track Analysis (NTA) Nanosight LM10 (Malvern Panalytical, United Kingdom).
EVs were characterized by flow cytometry using specific antibodies: anti-CD9 antibody (FITC) (Abcam, United Kingdom), anti-CD63 (APC) (Bio-Rad, USA) and anti-CD81 antibody (PE) (Bio-Rad, USA). Also, the Molecular Probes™ CellTrace™ Calcein Violet, AM (Invitrogen, USA) was used to ensure EVs integrity. The fluorescence was monitored by a Cytoflex S Flow Cytometer using a Violet Laser (405 nm) and its light Side Scattering (SSCviolet).