α bungarotoxin α bgt

Cultures were treated with lipopolysaccharide (LPS) (1 µg/mL) and ATP (5 mM) (Sigma-Aldrich, Madrid, Spain) to create a model of NLRP3 inflammasome activation. Pharmacological treatments were melatonin (10 nM), α-bungarotoxin (α-bgt, 100 nM) and luzindole (1 µM) (Sigma-Aldrich, Madrid, Spain). The compounds used to assess autophagy were chloroquine (inhibitor; Sigma-Aldrich, Madrid, Spain) and rapamycin (inductor; Sigma-Aldrich, Madrid, Spain).

Cells were suspended in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in a 5% CO₂ atmosphere. The PBMCs (1×10⁶ cells/ml) were cultured for 72 h in 24-well plates and subsequently stimulated with anti-CD3 (3 μg/ml, clone HIT3a; BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD28 (3 μg/ml, clone CD28.2; BD Biosciences) antibodies in the presence or absence of different concentrations (0.01, 0.1 or 1 μmol/l) of GTS-21 (Abcam, Cambridge, MA, USA). Purified CD4⁺ T cells (1×10⁶ cells/ml) were stimulated using anti-CD3-coated 96-well plates (BioCoat™ anti-human CD3 T-cell activation plates; BD Biosciences) plus anti-CD28 antibodies (3 μg/ml), in the presence of IL-12 (15 ng/ml, Peprotech, Inc., Rocky Hill, NJ, USA) and anti-IL-4 antibodies (4 μg/ml, Peprotech, Inc.) for Th1 differentiation for 72 h with GTS-21 (1 μmol/l) alone or combined with α-bungarotoxin (αBgt, 1 μmol/l; Sigma, St. Louis, MO, USA).