Caco 2 tc7 cells

Caco-2 TC7 cells (ATCC) were seeded at 7.5 x10⁴/ml and upon reaching confluence (7 days) the medium was changed every day for 7 the following 7 days. Before infection the cells were starved for 3 h in serum free DMEM. Monolayers were infected with primed EPEC(20 (link)) at an MOI of 1:10 for 3 h. The cells were then washed twice in PBS and the medium was replaced with serum free DMEM-high glucose plus penicillin and streptomycin at 100 U/ml and 100 μg/ml, respectively. After 1 h cells were washed and either processed for Western Blot (total IL-18) or incubated for a further 17 h (secreted caspase-4 and IL-18) with or without Ac-LEVD-CHO (Enzo Lifesciences). Supernatants were collected, cleared by centrifugation at 13000 rpm at 4 °C for 10 min and precipitated for Western blotting with the addition of 10 % (v/v) trichloroacetic acid for 17 h at 4 ºC. The concentration of IL-18 in cell supernatant (MBL) was determined by ELISA according to the manufacturer’s protocol.