Z yvad fmk

In the in vitro inhibiting experiment, LPS-primed PMs were pre-incubated with the following inhibitors: KCl (50 mM, PB0440, Sangon Biotech) to block K⁺ efflux, ATP receptor P2X7R inhibitor oxidized ATP (oATP, 500 μM, A6779, Sigma-Aldrich), cathepsin B inhibitor CA-074Me (10 μM, C5857, Calbiochem), Nlrp3 inhibitors including MCC950 (10 μM, S7809, Selleckchem), CY-09 (5 μM, S5774, Selleckchem) and Oridomin (20 μM, S2335, Selleckchem), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707, Alexis Biochemicals), at the indicated concentrations for 1 h. Then, LPS-primed PMs were treated with the Stx2 for 16 h in vitro. All supernatants were collected and detected for IL-1β by ELISA and LDH with Cytotox 96 Kit.

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).