Human il 8 cxcl8 elisa kit

After 24 h of treatment, the culture medium was collected from individual wells and frozen at -80°C for subsequent pro-inflammatory cytokine release determination. Culture medium was analyzed for the presence of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) using commercially available quantitative ELISA assay kits (Human IL-6 ELISA Kit, Human IL-8 / CXCL8 ELISA Kit, Human Tumor Necrosis Factor α ELISA Kit, Sigma-Aldrich Chemical Company, St. Louis, MO, USA) and following the manufacturer's instruction. Optical densities were read at 450 nm using an ELISA reader (Multiskan GO microplate spectrophotometer, Thermo Scientific, Waltham, MA, USA).

For this assay, cells were exposed for 11 days with Hg(II) or MeHg as described in section 2.4. After exposure, apical and basolateral media were recovered for analysis of the pro-inflammatory cytokines IL-8 and IL-1β using Human IL-8/CXCL8 ELISA kit (Sigma) and Human IL-1β ELISA kit (Sigma), following the manufacturer instructions. To determine the influence of macrophages in the pro-inflammatory response of intestinal cells, a parallel exposure assay with Hg(II) or MeHg was performed using monolayers of Caco-2/HT29-MTX without the incorporation of differentiated THP-1 cells into the basolateral compartment. The conditions of seeding and Hg treatment were identical to those described above. The identification of the inflammatory pathways triggered by Hg was carried out using inhibitors. Cells were seeded as described above, Hg treatments were applied to the apical side, while inhibitors were added to the basolateral medium during the duration of the exposure, changing media every 2 days. The inhibitors used were: SP600125 (300 nM), BIRB796 (300 nM), BAY 11-7082 (250 nM) (MedChemExpress®). After exposure (6 days), cells were collected to analyse the gene

Cells were seeded at a density of 3 Â 10 4 cells per cm 2 in 12-well plates. Nine days after seeding, the monolayers were exposed for 2, 4 and 24 h to As(III) (1 and 3 mg L À1 ) and As(V) (5 and 8 mg L À1 ) prepared in MEMc. When the exposure time ended, the medium was recovered for analysis of the pro-inflammatory cytokines IL-2, IL-6, IL-8 and TNFa, using specific ELISA kits [IL-2 Human Instant ELISAt Kit (Invitrogen, Fisher Scientific), Human IL-6 Elisa kit (Sigma), Human IL-8/CXCL8 Elisa Kit (Sigma), and TNF alpha Human ELISA Kit (Invitrogen, Fisher Scientific)], following the manufacturers' instructions. The values were normalized per mg of protein determined by the Bradford method (Bio-Rad Protein Assay, Bio-Rad, Hercules, CS, USA).