DMEM Selleck LY2606368 medium was supplemented with the same concentration of L-tyrosine and agmatine sulphate as used for the gastrointestinal

experiments. In the adhesion assay experiments, bacteria grown in MRS to the mid-exponential phase (OD620 = 0.8) as for BA induction, were centrifuged (10.000 x g, 10 min), washed once with cold phosphate-buffered saline (PBS) pH 7.1 (10 mM Na2HPO4, 1 mM KH2PO4, 140 mM NaCl, 3 mM KCl, all purchased from Merck, Darmstadt, Germany) and resuspended in the same DMEM medium supplemented, or not, with tyrosine, agmatine or both. Bacterial suspensions were added to Caco-2 intestinal cells in a final selleck chemical volume of 0.1 mL and a final concentration of 1.25 x 107 CFU mL-1 (ratio 1:100, Caco-2 cells to bacteria) and incubated at 37°C for 1 h. Unbound bacteria were then removed by washing three times with 0.2 mL of PBS at pH 7.1. Some wells, unwashed, were used as control. Cell cultures were then resuspended in 0.1 mL of PBS and detached by adding 0.1 ml of 0.05% trypsin-EDTA (Gibco, Carlsbad, CA). After incubation at 37°C for 10 min, the detachment reaction was interrupted by adding 0.1 mL of cold PBS. The number of total and adhered bacteria was determined by serial dilution and quantitation on agar plates as for viable counts. The adhesion percentage was calculated by comparing the number of CFU from three washed wells with those from control wells. Every experiment was performed in

triplicate. RP-HPLC determination of BA Pre-column dabsyl chloride manual derivatisation was performed for BA detection. The derivatisation https://www.selleckchem.com/products/incb28060.html reaction was carried out as described by Krause et al. [40]. 10 μl of the dabsylated supernatants were used for injection. HPLC analysis was performed using an Alliance 2795 system (Waters, Milford, MA) equipped

with a Waters Nova-Pack C18 column (150 × 3.9 mm 4 μm particle size). Dabsylated amino acids and amines were eluted using the gradient described by Krause et al. [40]. Detection was carried out by a Waters 2996 Photodiode array detector at 436 nm. RNA extraction and Real Time PCR analysis Transcriptional analysis was performed after 20 min gastric stress simulation. Control and samples mimicking gastric stress at pH 5.0, were analyzed in the presence or absence of biogenic amine precursors. Total RNAs were extracted pheromone from 2 × 109 cells using the FastRNA pro blue kit (Qbiogene, Montreal, QC) following the manufacturer’s instructions. Cells were lysed mechanically with a Hybaid Ribolyser for 30 s. The RNAs’ quantity and quality was determined by spectrophotometry, and their integrity was assessed by visualization of the rRNA bands on 1.2% agarose gels. Absence of chromosomal DNA was confirmed by quantitative real-time PCR. cDNAs were synthesized using 0.8 μg of total RNA and Quantitect Reverse Transcription (Qiagen, Hilden, Germany) which included a DNase treatment and reverse transcription.