Human mini elisa kits

Growth factors were detected in EV samples through commercially available kits, following manufacturer protocols: Human TGF-β1 Platinum ELISA kit (eBioscience, UK); PDGF-BB and FGF-2 using respective Human Mini ELISA kits (PeproTech, UK); Human IGF-1 Quantikine® ELISA kit (R&D Systems, UK). Assays were performed in duplicate on three separate occasions.

THP-1 cells were treated with PM10 in the absence and presence of test materials for 72 h in the serum-free culture medium. The conditioned medium was collected and the concentrations of TNF-α, IL-1β, MCP-1, and ICAM-1 were measured using Human Mini ELISA kits (PeproTech, Rocky Hill, NJ, USA), according to the manufacturer's instructions. Briefly, samples (100 μL conditioned medium) or solutions of standard at varied concentrations were added to microplate wells which contained immobilized capture antibody. After 12 h incubation at 4°C, the wells were washed and solutions of biotinylated detection antibody were added and incubated for 2 h. After washing the wells, solutions of horseradish peroxidase conjugated to streptavidin or avidin were added and incubated for 30 min. The cells were washed, and 3,3′,5,5′-tetramethylbenzidine (TMB) or 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrate solution was added to the wells to initiate enzymatic color development. The TMB reaction was terminated using 1 M HCl and the absorbance of the reaction mixture was measured at 450 nm. The absorbance of the ABTS reaction mixture was measured at 405 nm.