Recombinant human il 8 protein

The IL-8 protein level in the culture medium secreted by Sc and Gi HepG2 cells with or without palmitate treatment was quantified by the human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (R&D systems). In brief, 3×10⁶ HepG2 cells were cultured in petridish for overnight followed by treating with or without palmitate for 24 hr at 37°C. The culture medium of each sample was collected and concentrated with 5 kDa centrifugal filter units (Millipore). The concentrated medium of each sample and IL-8 standard (50 μl) were transferred to the wells containing 100 μl assay diluent in a flat-bottomed 96-well plate coated with IL-8 antibody and incubated for 2 hr at room temperature. The plate was aspirated and washed followed by adding 100 μl IL-8 conjugate (IL-8 antibody conjugated to HRP) for 1 hr at room temperature. The plate was then aspirated and washed for four times. The working solution of Streptavidin-HRP was added subsequently and incubated for 30 min at room temperature followed by aspiration. Lastly, the stabilized chromogen was added to the plate for 30 min at room temperature in the dark followed by adding stop solution. The absorbance of each well was detected at 450 nm with a reference at 490 nm in a microplate reader (Versa MAX, Molecular Devices). The recombinant human IL-8 protein (R&D systems) was used for the generation of a standard curve for IL-8.

Nec-1, GSK’872, NSA, SP600125, reparixin, Z-vad-fmk and Liprosxtatin-1 were bought from Selleck Chemicals (Houston, TX, USA). Anti-IL-8 antibody and recombinant human IL-8 Protein were bought from R&D Systems.