5 M NaCl and precipitated with 10 vol ethanol in the refrigerator overnight, then centrifuged at 20,000 × g for 20 min at 10°C and air dried. Purified Selleckchem Seliciclib LPS samples were redissolved in Laemmli sample buffer [49] at 95°C for 5 min. Samples were applied to 15% polyacrylamide/0.9% bis minigels containing 3.2 M urea with the Laemmli discontinuous buffer formulation [49], and a

5% stacking gel. After electrophoresis at 150 V for 75 min, gels were either fixed overnight for silver staining [50] or transferred to polyvinylidenedifluoride membrane using Tris/glycine transfer buffer [51]. Blots were blocked overnight in 3% bovine serum albumin and 0.03% NaN3 in the wash buffer described above for ELISA. Primary antibody (anti-Lewis X or anti-Lewis Y, 1:200) and secondary antibody (peroxidase-conjugated goat anti-mouse IgM, 1:1000) were Vadimezan cost diluted in wash buffer selleck containing 0.5% BSA. Colorimetric detection used 3,3′-diaminobenzidine with cobalt enhancement [52]. Densitometry was performed with the public domain application Image J, available at http://​rsb.​info.​nih.​gov/​ij. Results Little is known about the physiologic roles of cholesterol in H. pylori. To investigate responses of H. pylori to cholesterol, we adopted a defined, serum-free culture medium, F12 with 1 mg/ml albumin, in which this bacterium may be stably

passaged [26]. This modest concentration of albumin boosts growth [25, 26] and alleviates the tight adherence to culture surfaces that occurs in protein-free media [53]. In this defined medium, addition of 50 μg/ml cholesterol did not significantly alter the growth rate (Figure 2). The absence of growth effects under the chosen culture conditions was advantageous for investigation of the physiological importance of cholesterol in H. pylori. Thus, we were able to compare gastric colonization of gerbils by strain SS1 that had been cultured in the defined medium containing varied amounts of cholesterol (Figure 3). Eleven days after oral inoculation, H. pylori in gastric antrum were selectively plated and quantitated. Strikingly, gerbils were colonized only by the cultures grown in cholesterol-containing medium,

but not by H. pylori grown in cholesterol-free medium ADAMTS5 (In each experiment, P < .0001 for comparison of log (CFU/g) between groups using Student two-tailed t-test). Therefore, cholesterol was an essential component of the growth medium in order to establish H. pylori infection in this animal model. Figure 2 Addition of cholesterol to the defined medium does not affect H. pylori growth rate. Parallel cultures of each strain were grown overnight in F12/albumin (1 mg/ml) in the absence (open bars) or presence (shaded bars) of 50 μg/ml cholesterol. The initial population density was 2 × 106/ml. Doubling times were calculated from the measured increase in biomass. Values shown represent the mean ± sd of five or more independent measurements. n. s.