Quantifying CXCL1 and CXCR2 Levels in Spinal Cord

An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of CXCL1 (Abcam, UK), and CXCR2 (Wuhan Fine Biotech Co., Wuhan, China) in the L4-5 levels of left spinal cord [24 (link)]. Spinal cord tissues were homogenized in a lysis buffer containing protease and phosphatase inhibitors. Tissue samples were centrifuged at 12,500× g for 10 min and the supernatant was collected. BCA Protein Assay (Pierce) was employed to determine protein concentrations. For each reaction in a 96-well plate, 100 μg of proteins from the samples was used. All ELISA experiments followed the manufacturer’s protocol. The optical densities of samples were measured using an ELISA plate reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 450 nm and the levels of CXCL21 and CXCR2 were calculated using the standard curves and normalized to the total protein levels.