Bioluminescence assay kit

IL-1β and IL-6 concentrations in cell culture supernatants and/or CSF samples were determined by ELISA (R&D Systems, Germany), according to the manufacturer’s instructions. ATP was indirectly measured with a bioluminescence assay kit (Molecular Probes, Oregon, USA).

Various cells were incubated with WT, Δvfh, or Δvfh containing pUC-vfh or control vector pUC18 strains of V. fluvialis at a MOI of 50 per cell for 3 h without LPS priming. For some experiments, the BMMs were pre-incubated for 2 h with CA-074 Me (C5857, Calbiochem, San Diego, CA, USA), Z-YVAD-FMK (Alexis Biochemicals, Lörrach, Germany), oATP (Merck, Darmstadt, Germany), N-acetyl-L-cysteine (NAC; A7250 Sigma-Aldrich), or KCl at the indicated concentrations. At the indicated time points, LDH activity in the culture supernatants was measured using a Cytotox96 Kit (Promega, Madison, WI, USA). The concentrations of cytokines in cell-free supernatants were quantified using commercial ELISA kits (BD Biosciences, San Jose, CA, USA). The release of ATP from the BMMs was monitored using a bioluminescence assay kit (Molecular Probes).