Competitive enzyme linked immunosorbent assay kit

Immature DCs (10⁶/mL) were challenged with Pb18 or Pb265 (2 x 10⁵ yeasts/mL) using a DCs/yeasts ratio of 5:1, or treated with LPS (5 μg/mL) for 1, 2, 4, 8, 12, 18, 24 or 48 h. Supernatants were harvested and assayed for PGE₂ levels using a competitive enzyme-linked immunosorbent assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). In some experiments, DCs were incubated for 2h with monoclonal antibodies: anti-TLR2 (2 μg/10⁶ cells), and/or anti-MR (2 μg/10⁶ cells) (monoclonal antibodies purchased from Biolegend, San Diego, CA, USA), anti-dectin-1 (3 μg/10⁶ cells) and anti-DC-SIGN (4 μg/10⁶ cells) before fungus challenge (monoclonal antibodies purchased from R&D Systems, Minneapolis, MN, USA). These concentrations were chosen because they induced the highest percentages of blockage in previous experiments (data not shown). The protocols were designed in order to block three receptors keeping only one available.

The level of 8-OHdG and 8-oxo-Gua was measured in 250 ng total nucleic acids extracted from left ventricular cardiomyocytes using a competitive enzyme-linked immunosorbent assay kit (Cayman Chemical, catalog no. 589320) according to the manufacturer’s instructions. The samples were incubated for 1 h with a monoclonal antibody against 8-OHdG in a microtiter plate precoated with 8-OHdG and 8-oxo-Gua. The final color was developed by the addition of 3,3,5,5-tetramethylbenzidine, and absorbance was measured at 450 nm. The samples were diluted at 1:50 with enzyme immunoassay buffer before assaying.