In some experiments, astrocytes were incubated with a medium containing Z-YVAD-FMK (10 μM), a potent cell permeable and irreversible caspase-1 inhibitor (ICE; Parajuli et al., 2013; Tseng et al., 2013) or with Z-VAD-FMK (20 μM), a pan-caspase inhibitor (Dostert et al., 2009) or NLRP3 blocking peptide (bp; 4 μg/ml; Abcam) or Mito-TEMPO (500 μM), a ROS scavenger, for 30 or 60 min (for Mito-TEMPO) before and during ethanol (50 mM), or with ATP (5 mM) or LPS (50 ng/ml) treatments. Caspase-1 enzymatic activity, ROS release, IL-1β and IL-18 cytokines were determined in cells after 24 h treatments. Sterile toxin-free culture materials were used for all the experiments. In addition, the possible contamination of ethanol with LPS was determined by using the chromogenic limulus amebocyte lysate test, following the manufacturer’s instructions (Lonza Verviers SPRL). The endotoxin content in ethanol solution was 2.98 × 103 pg/ml, which is far below the concentration required to induce astroglial activation under our assay conditions, as previously described (Alfonso-Loeches et al., 2010).
