5/OD of growth) [24]. Adherence to Caco-2 cells Adherence to Caco-2 cells was investigated using methods described previously [28]. In brief, cells were cultivated in DMEM medium supplemented with 10% fetal bovine serum and 1% non-essential

amino acids under a 5% CO2 atmosphere. All the experiments were performed on cells between the 15th and 25th passage. Caco-2 cells were cultivated in 24-well plates to a density of 1 × 105 cells/well for 3-5 days. Bacteria were grown to mid-log phase at 37°C without agitation in tryptic soy broth; Caco-2 cells were incubated with bacteria for 2 h at a multiplicity of infection of 100:1. After infection of the monolayer, epithelial cells were washed and lysed with 0.25% Triton-X at 37°C for 20 min and adherent bacteria enumerated by quantitative bacterial counts. Pilot experiments had shown no significant bacterial Selleck Epoxomicin invasion under the outlined

conditions. Isolation and analysis of glycolipids and LTA Bacterial cells were resuspended in 0.1 M citrate buffer pH 4.7 and cell walls disrupted by shaking with an equal volume of glass beads (0.1 mm glass beads, 3 × 1 min intervals using a BeadBeater, Glenn Mills, Clifton, NJ). Glass beads were removed by sedimentation, and disrupted cells were stirred with an equal volume of n-butanol for 30 min. After phase separation by centrifugation, the www.selleckchem.com/products/blz945.html aqueous layer was removed, dialyzed against 0.1 M ammonium acetate (pH 4.7) and lyophilized. LTA was purified from the aqueous phase

by hydrophobic interaction chromatography [4]. The butanol phase was evaporated under a vacuum, and cell membrane lipids were extracted according to the method of Bligh and Dyer and separated by TLC (0.2 mm Silica gel 60 F254 Merck, Darmstadt) using a solvent system of CHCl3/MeOH/H2O (65:25:4, v/v/v) and detection with α-naphthol (3.2%). For detection of phospholipids, TLC plates were stained with molybdenum blue; amino phospholipids were stained with ninhydrin, as previously described [29]. LTA was also analyzed by SDS-PAGE as described previously [5]. Briefly, bacterial cell walls were disrupted by shaking with glass beads as described above, boiled in sample buffer AC220 in vitro containing SDS, and subjected to SDS-PAGE in gradient gels containing acrylamide (4/12% w/v, Invitrogen). Separated LTA was transferred onto PVDF RVX-208 membrane and blocked at 4°C in Tris-buffered saline (TBS) containing skim milk (5% w/v) for 18 h, then incubated at 20-22°C for 2 h with rabbit antibody raised against E. faecalis LTA (see below) diluted 1:200 in TBS/skim milk. After washing in TTBS (Tween 20 0.05% v/v in TBS), the sheets were incubated at 20-22°C for 1 h with a goat anti-rabbit IgG (whole cell) alkaline phosphatase conjugate (Sigma), diluted 1:1000 with TBS/skim milk, and then washed again in TTBS. Binding of the enzyme-conjugated antibodies was detected with the NBI/BCIP (Biorad). For visualization of proteins, SDS PAGE gels were stained with Coomassie blue.