Quantikine elisa human cxcl1 gro α immunoassay

Plasma CXCL1 [growth-regulated oncogene (GRO)-α] concentrations were analyzed using the Quantikine ELISA human CXCL1/GRO-α immunoassay (R&D Systems) as per the manufacturer's instructions. Assay diluent (RD1U) was added to each well, followed by 200 μL of standard, control, or plasma samples before incubating at room temperature for 2 h. Wells were aspirated, washed thrice before addition of human GRO-α conjugate, and incubated for 2 h at 2–4°C. The aspiration and washing steps were repeated, 200 μL of substrate solution was added, and the ELISA plate was incubated for 30 mins at room temperature in the dark. After addition of 50 μL stop solution, optical density was measured at 450 nm with wavelength correction at 540 nm, using a Fluostar Optima plate reader (BMG Biotech).

The collected blood was centrifuged according to our standard protocol at 3000 rpm for 10 minutes at 4 degrees Celsius, 30 min to one hour after sampling. The serum was stored at −80 degrees for further processing. The serum sCD40 Ligand and CXCL1 (GROα) concentrations were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers' protocols. All experiments were performed in duplicate. The sCD40L-serum levels were measured using the commercially available human sCD40L Instant ELISA (Cat. number BMS239INST human sCD40L, eBioscience). Concentrations were measured in ng/mL. The CXCL1 serum levels were measured using the commercially available human CXCL1/GRO alpha Quantikine ELISA Kit in concentrations as pg/mL (Cat. number DGR00 Quantikine ELISA Human CXCL1/GROα Immunoassay, R&D Systems).