Solid white bottom

Caspase 3/7 activity was measured by using a Caspase-Glo assay kit (Promega, G8091) following the manufacturer’s instructions. EGCs (10⁴ cells/well) seeded in a white tissue culture-treated 96-well plates (Falcon, solid white bottom) were treated with TcdA or TcdB alone or in the presence with 10Panx (50µM) or A438079 (300µM) for 18 h. Then, the plates containing the cells were removed from the incubator for 30 min. A volume of 100 µL of Caspase-Glo reagent was added to each well, and the wells were mixed with a plate shaker at 500 rpm for 30 s. The plates were incubated for 2 h at room temperature. Then, the luminescence of each sample was acquired in a plate-reading luminometer (Promega) to obtain the relative luminescent units (RLUs) subtracted by the background.

Caspase 3/7 activity was evaluated with a Caspase-Glo assay kit (Promega, G8091) following the manufacturer’s instructions. EGCs (10⁴ cells/well) were seeded in white tissue culture-treated 96-well plates (Falcon, solid white bottom) and treated with TcdA or TcdB for 18 h in the presence or absence 300 µM ATL313 (an A2a-selective adenosine receptor agonist), 100 µM ATL801 (an A2b-selective adenosine receptor antagonist) or 30 µM CI-IBMECA (an A3AR-selective adenosine receptor agonist) added one hour prior to C. difficile toxin challenge. Then, the plates containing the cells were removed from the incubator for 15 min. A volume of 100 µL of Caspase-Glo reagent was added to each well, and the wells were mixed with a plate shaker at 500 rpm for 30 s. The plates were incubated for 2 h at room temperature. Then, the luminescence of each sample was acquired in a multi-mode reader (Synergy/HTX, Biotek) to obtain the relative luminescent units (RLUs).

To measure the levels of cyclic adenosine monophosphate (cAMP) in EGCs treated with TcdA or TcdB for 18 h, the cAMP-Glo assay (Promega, V1501) was performed following the manufacturer’s instructions. The assay is based on the principle that cAMP stimulates protein kinase A (PKA) holoenzyme activity, decreasing available ATP and leading to decreased light production in a coupled luciferase reaction. Briefly, EGCs (10⁴ cells/well) were seeded in white tissue culture-treated 96-well plates (Falcon, solid white bottom) and treated with TcdA or TcdB for 18 h. Then, after removal of the supernatant, cells were incubated with 20 µL of cAMP-Glo lysis buffer with shaking at room temperature for 20 min. After this, 40 µL of cAMP-Glo detection solution (2.5 µL of PKA in 1000 µL cAMP-Glo reaction buffer) were added to each well and the plates were shaken for 60s and incubated at room temperature for 20 min. Then, 80 µL of kinase-Glo reagent were added to each well, and the plates were shaken for 60 s and incubated at room temperature for 10 min. The luminescence was recorded using a multi-mode reader (Synergy/HTX, Biotek), and the intrinsic reagent luminescence (no-cell, no-compound background control) was subtracted from the luminescence signals in the sample to obtain the relative luminescent units (RLUs).