The coverslips were examined using a light microscope to determine the adherence of
the strains. Categories (+++ to −) were assigned through comparison of the size of the microcolonies. Contact hemolysis assay was performed as previously described with slight modification (32). Bacterial strains were grown overnight in BHI broth at 30 C. The cultures were then diluted 1:100 into 5 ml of DMEM and shaken at 250 rpm for 100 min in 15-ml conical polypropylene tubes at 37 C. Next, the bacterial aggregates were centrifuged at 2,000 × g for 15 min. The pellets were resuspended into 5 ml of DMEM, 50 μl of which was placed onto 96-well microtiter plates and monitored for viable bacteria at an optical wavelength of Dactolisib research buy 600 nm. 50 μl of a 25% sheep RBC (Nippon Biotest Laboratories Inc., Tokyo, Japan)-DMEM suspension was added and this was centrifuged at 1,000 × g for 15 min to form a close EPEC-RBC contact. After 2 hr of incubation at 37 C, CP-868596 in vivo bacterium-RBC pellets were gently resuspended to facilitate the release of hemoglobin. Cells were centrifuged at 1,000 × g for 15 min, and the supernatant was monitored for released hemoglobin at an optical wavelength of 550 nm. Similarly-treated uninfected RBCs were used as a spectrophotometric zero. The hemolysis ratio was calculated using E2348/69 as a standard.
RT-PCR was used to analyse the transcriptional expression of the bfpA gene indicating expression of the bundlin. Overnight
bacteria cultures were diluted 1:100 in DMEM F-12 broth and grown to the mid-logarithmic phase (OD600= 0.5) at 37 C with shaking. Cultures were pelleted by centrifugation at 13,000 × g for 10 min, and RNA was isolated using TRIzol® Reagent (Invitrogen, Faraday Avenue, CA, USA) according to the manufacturer’s instructions. Total RNA (1 μg) and 60 μM of random hexamers (Roche, Mannheim, Germany) were incubated for 10 min at 65 C, immediately cooled on ice and then reverse transcribed in a final volume of 20 μl-containing 1 mM of deoxynucleotide mix, 20 U of RNase inhibitor, Transcriptor RT reaction Tau-protein kinase Buffer 1× and 10 U of Transcriptor reverse transcriptase (Roche)-that was reacted for 30 min at 55 C. PCR amplification of cDNA was performed with an initial denaturation step of 5 min at 94 C, followed by 19 cycles of 30 sec at 94 C, 1 min at 55 C and 1.5 min at 72 C, and finishing with one cycle of 10 min at 72 C, using primer sets for the bfpA gene (Table 1). The number of PCR cycles used came within the linearity range for PCR amplification and constitutive expression of 16S rRNA assessed from the same cDNA preparation was used as a standard. Samples (10 μl) of each PCR product were separated by electrophoresis in 2.0% agarose and visualized by ethidium bromide staining. The bands of the bfpA gene were confirmed visually and results were standardized with the 16S rRNA band density.