Human il 8 elisa max deluxe

The amount of IL-8, which is an inflammatory cytokine, in HEp-2 cell culture supernatant was quantified using a commercial kit, Human IL-8 ELISA MAX™ Deluxe (BioLegend, Tokyo, Japan), according to the manufacturer’s protocol. The level of IL-8 gene expression was also determined by qRT-PCR using primer sets specific to IL-8 and an internal control (gapdh: glyceraldehyde-3-phosphate dehydrogenase) as described previously [16 (link)].

AGS cells were seeded onto 6-well cell culture plates at a density of 4 × 10⁵ cells per well and were then incubated for 2 days at 37°C. At 2 h prior to infection, cells were washed with PBS and the medium was changed to 2 ml of RPMI 1640 containing 2% FBS and no antibiotics. Liquid cultures of H. pylori were resuspended in RPMI 1640 containing 2% FBS, and AGS cells were infected at a MOI of 10; the lower MOI, compared to that used for the cell elongation assays, was used to prevent significant levels of cell death during the longer incubation required in the IL-8 assay. The infection was allowed to proceed for 5 h as a means to measure cag PAI-dependent immediate IL-8 secretion and for 30 h as a means to measure CagA-dependent late IL-8 secretion (34 (link)). At 5 and 30 h postinfection, 1 ml of cell culture medium was taken and centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was used for IL-8 enzyme-linked immunosorbent assay (ELISA). Assays were performed using human IL-8 ELISA Max Deluxe (BioLegend, San Diego, CA) following the manufacturer’s instructions, and absorbance was measured using an Epoch microplate spectrophotometer (BioTek, Winooski, VT). Data were presented as means ± SD of results from 3 independent replicates.

Transfection was performed as described in paragraph ‘Transfection’ and stimulation as described in paragraph’ Stimulation with Bile Acids’. HepG2 cells were washed with PBS, lysed in the RIPA buffer and the protein concentration was measured with the BCA Protein Assay Macro Kit according to the manufacturer’s protocol (Serva Gelelectrophoresis GmbH). One hundred microliters of the total lysate was used for the IL-8 enzyme-linked immunosorbent assay (ELISA). The assay was performed using human IL-8 ELISA Max Deluxe (BioLegend) following manufacturer’s instructions. Absorbance was measured at 450 and 570 nm using a plate reader (EnSpire, Perkin Elmer).