Triton x 100

The bacterial adhesion assay was performed as described previously (Boudeau et al., 1999 (link)). Briefly, Intestine-407 cells were seeded in 24-well tissue culture plates with 4 × 10⁵ cells per well. Monolayers were then infected at a multiplicity of infection of 10 bacteria per cell in 1 mL of the cell culture medium without antibiotics and with heat-inactivated fetal calf serum (FCS, PAA), using bacteria grown at 37°C in LB with or without CMC or P80 at various concentration (0.0625, 0.2500 and 1.0000) and subsequently washed twice in PBS in order to avoid any direct effect of emulsifier on adhesion and invasion processes. After a 3 h incubation period at 37°C, monolayers were washed three times in phosphate-buffered saline (PBS, pH 7.2). Epithelial cells were then lysed with 1% Triton X-100 (Euromedex) in deionized water. Samples were diluted and plated onto Muller-Hinton agar plates to determine the number of colony-forming units (CFU) corresponding to the total number of cell-associated bacteria (adherent and intracellular bacteria). In order to determine the number of intracellular bacteria, fresh cell culture medium containing 100 mg.ml^-1 gentamicin was added for 1h to kill extracellular bacteria. Monolayers were then lysed with 1% Triton X-100, and bacteria were quantified as described above.

10 million cells were cross-linked in medium with 1% formaldehyde for 8 min at RT on a slow shaker, quenched with freshly prepared 0.125M glycine, incubated 5min at RT on a slow shaker, then pelleted at 400 g for 5 minutes at 4°C, washed three times with 30ml of ice cold PBS and then incubated for 20 minutes rotating at 4°C in 1mL of RIPA lysis buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0 (Invitrogen), 140mM NaCl, 1% (v/v) Triton X-100 (Euromedex), 0.1% (v/v) SDS and 0.1% sodium deoxycholate (SIGMA)). Nuclei were pelleted at 1350 g for 5 minutes at 4°C, washed for 10 minutes rotating with 1ml of a buffer containing 10mM Tris, 200mM NaCl, 1mM EDTA (Invitrogen), 0.5 mM EGTA (Euromedex), pelleted and lysed in buffer containing 0.4% SDS (Euromedex), 10mM EDTA (Invitrogen), 50mM Tris-HCl pH 8.0 for 30min on ice (volume of buffer = 100μl/1.6 million cells). Lysates were sonicated on a Bioruptor Pico (Diagenode) sonication devices (11cycles 30 s ON, 30 s OFF) to reach fragments ranging from 150 to 500bp, and then centrifuged at 10,000 g for 10 minutes at 4°C to remove debris. Samples were then snap-frozen in liquid nitrogen and stored at −80°C until immunoprecipitation. All buffers contained cOmplete EDTA free Protease inhibitor cocktail (Roche).