Pfa solution

EGC lines (4×10⁴ cells/well) plated on eight-chamber glass tissue culture slides in a polystyrene vessel and treated with TcdA or TcdB for 18 h were fixed in 4% PFA solution (Alfa Aesar) in PBS for 30 min at room temperature and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) and 3% bovine serum albumin (BSA, Sigma) in PBS for 10 min at 4°C. After blocking with 5% normal donkey serum (Jackson ImmunoResearch, 017-000-121) in PBS for 30 min at room temperature, the cells were incubated with anti-NFκBp65 (Santa Cruz Biotechnology, sc-372) overnight at 4°C. After three washes with washing buffer (0.01% Tween 20 in PBS), the cells were incubated for 1 h with Cy3-conjugated AffiniPure donkey anti-rabbit (Jackson ImmunoResearch, 711-165-152) antibody, washed with PBS, and mounted with ProLong Gold antifade reagent containing DAPI (Thermo Scientific, P36931). The samples were visualized by fluorescence microscopy (Zeiss). For each experimental condition, 100 cells were counted, and the percentage of cells (%) with positive nuclear staining for NFκBp65 was determined.

EGCs (4x10⁴ cells/well) plated on 4-chamber glass tissue culture slides in a polystyrene vessel and treated with TcdA or TcdB for 18 h were fixed in 4% PFA solution (Alfa Aesar) in PBS for 30 min at room temperature and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) and 3% bovine serum albumin (BSA, Sigma) in PBS for 10 min at 4°C. After blocking with 5% normal donkey serum (Jackson ImmunoResearch, 017-000-121) in PBS for 30 min at room temperature, the cells were incubated with anti-A2A (Invitrogen, PA1-042, 1:50), anti-A2B (Invitrogen, PA5-72850, 1:100), anti-A3 (Invitrogen, PA5-36350, 1:50) or anti-IL-6 (1:20, R&D system, AF506) overnight at 4°C. After three washes with washing buffer (0.01% Tween 20 in PBS), the cells were incubated for 2 h with Alexa fluor 594-conjugated donkey anti-rabbit (Abcam, ab150064, 1:400), Alexa fluor 488-conjugated donkey anti-rabbit (Abcam, ab150073, 1:400) or Alexa fluor 488-conjugated donkey anti-goat (Abcam, ab150129, 1:400) antibodies, washed with PBS and mounted with ProLong Gold antifade reagent containing DAPI (Thermo Scientific, P36931). The samples were visualized by fluorescence microscopy (Evos, Thermo Scientific). The intensity of fluorescence was measured using Image J software.

EGCs line (4x10⁴ cells/well) plated on 8-chamber glass tissue culture slides in a polystyrene vessel and treated with TcdA or TcdB for 18 h were fixed in 4% PFA solution (Alfa Aesar) in PBS for 30 min at room temperature and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) and 3% bovine serum albumin (BSA, Sigma) in PBS for 10 min at 4°C. After blocking with 5% normal bovine serum albumin in PBS for 40 min at room temperature, the cells were incubated with anti-Panx-1 antibody (1:100, Invitrogen, 488100), anti-P2X7 antibody (1:50, Millipore, AB5246), anti-IL-6 (1:20, R&D system, AF506) or phosphorylated NFκB (1:100, Thermo Scientific, PA537718) overnight at 4°C. After three washes with washing buffer (0.01% Tween 20 in PBS), the cells were incubated for 2h with secondary antibody conjugated with Alexa Fluor 488 or 594 (1:400, Invitrogen, A21206/abcam, ab150129/abcam, ab150064) at room temperature, washed with PBS and mounted with ProLong Gold antifade reagent containing DAPI (Thermo Scientific, P36931). The samples were visualized by fluorescence microscopy (Zeiss).