Exoglow protein ev labeling kit red

Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose, L-glutamine, sodium pyruvate and sodium bicarbonate, Hank’s Balanced Salt Solution (HBSS) modified with sodium bicarbonate (without phenol red), non-essential amino acids (NEAA), antibiotic/antimycotic solution (penicillin, streptomycin, and amphotericin) and foetal bovine serum (FBS, non-USA origin), Curcumin (CUR), Triton X-100 and QuantiPro^TM BCA Assay Kit were purchased from Sigma-Aldrich (Poole, UK). Caco-2 cells were purchased from the European Collection of Cell Cultures (ECACC) and used between passages p66–p74. MTS reagent, namely 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (commercially known as CellTiter 96 AQueous One Solution Assay) was purchased from Promega (Madison, WI, USA). Exosome isolation kit from other body fluids and exosome isolation kit form cell culture were purchased from Thermo Fisher Scientific (Loughborough, UK), ExoGlow™-Protein EV Labeling Kit (Red) and exosome-depleted FBS were purchased from System Biosciences (Palo Alto, CA, USA). Transwell^® permeable cell culture inserts (polycarbonate filter, 1.1 cm² diameter, 0.4 μm pores) were obtained from Corning (Corning, NY, USA). PD-10 columns, Sephadex G-25 were obtained from GE Healthcare (Chicago, IL, USA).

Exosomes were labeled using the ExoGlow™‐Protein EV Labeling Kit (Red) (EXOGP100A‐1, System Bioscience) according to the manufacturer's instructions. The N2a cells cultured on sterile coverslips were incubated with the labeled exosomes for 24 hours and fixed with paraformaldehyde. Immunofluorescent staining was performed using NeuN antibody (AF1072, Beyotime) according to the instructions, and the stained cells were mounted with a drop of Antifade Mounting Medium containing DAPI (P0131, Beyotime). The internalized exosomes, N2a cells, and nucleus were, respectively, detected using 573 nm, 511 nm, and 364 nm excitation lasers.