Cxcr2

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.

Total RNA was extracted by Trizol reagent (Thermo), according to the manufacturer’s instructions after 24 h post-treatment. The total RNA concentration was determined in RNAase-free water using Nanodrop, while the concentration was determined using Qubit Fluorometer 3.0 (Thermo). 1 μg of total RNA was reverse transcribed using a 5X All-In-One RT MasterMix (Applied Biological Materials, Richmond, BC, Canada) into cDNA using thermo-block (Eppendorf). Finally, the real-time PCR was carried out on ABI 7300HT sequence detection system (ABI), containing 2X TaqMan Gene Expression Master Mix (Invitrogen, USA), DEPC water and 5 μL of cDNA and 1 μL the following primers: Prime Time qPCR Assay: GLUT4, CXCR2, IL-10, CXCL-1, TNFA, GLUT1 and GLUT4 qRnoCIP0027857 were purchased from Biorad, Des Plaines, IL, USA. Triplicate samples were run for each gene. The reference gene GAPDH qRnoCIP0050838 was applied as an internal control to normalize the expression of target genes. Relative expression levels were determined for each sample after normalization against the reference gene, using the ΔΔCt method to compare relative fold expression differences.