Next, 10 μL of either Quant-iT DNA BR standard solutions or test DNA samples were added to the

cell and mixed well. Fluorescence BI-6727 was measured using excitation/emission maxima ∼ 510/527 nm. The results of quantification were compared by the band intensity of the genomic DNA as visualized on ethidium bromide-stained 1% agarose gel following electrophoresis and the band intensity was calculated by using a Kodak Gel Logic 200 imaging system (Carestream Health, Rochester, NY, USA). Propidium monoazide and EMA (Biotium, Hayward, CA, USA) were dissolved in 20% DMSO (Sigma-Aldrich, St. Louis, MO, USA) to obtain a 20 mM stock solution. The PMA and EMA solutions were then diluted and added to the bacterial suspensions. Final concentrations of PMA were 0, 5, 10, 50, and 100 μM and of EMA 0, 1, 5, 10, and 50 μM. These were stored in a dark chamber at 4°C for 5 min, then the samples were exposed to light for 2 min using a 650 W halogen lamp (Philips Broadway, Suresnes, France) at a distance of 20 cm. During light exposure, the tubes were kept on ice to avoid excess heating. After the photo induced cross-linking process, the cells were pelleted at 12,000 rpm for 5 min before DNA extraction and isolation. Five hundred μL aliquots of cell suspension were stained for microscopic click here examination by the addition of 1.25 μL SYTO 9 (Invitrogen), 5 mM DMSO, and either 1.25 μL PMA or EMA (20 mM in 20% DMSO). SYTO 9 stains all

Calpain bacteria and PMA and EMA stain the bacteria if the dyes can penetrate the bacterial membranes. The samples were then mounted on microscope slides and photomicrographs taken 5 min after application of the dyes, using an Eclipse E800 microscope (Nikon, Tokyo, Japan)

with a 100 × /1.30 NA oil objective and FITC (excitation filter, 465–495 nm; dichroic mirror, 505 nm; absorption filter 515–555 nm)/TRITC (excitation filter, 540/25 nm; dichroic mirror, 565 nm; absorption filter 605/55 nm) fluorescence filter sets. The FITC filter was used for observing the SYTO 9 stained cells and the TRITC filter for the PMA or EMA stained ones. ACT-1 software version 2.63 (Nikon) was used for visualization. To quantify H. pylori, 1 μL of extracted genomic DNA was added to 49 μL of PCR mixture containing SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and 10 pmol of primers, sodB-F (5′-ATGTTTACATTACGAGAG-3′) and sodB-R (5′-TTAAGCTTTTTTATGCACC-3′) (24). The cycling conditions were 5 min at 95°C, followed by 40 cycles of 1 min at 95°C, 1 min at 43°C, and 1 min at 72°C. Then real-time PCR and data analysis were performed using a Real-Time PCR 7500 (Applied Biosystems). 7500 System Sequence Detection Software version 1.3.1 (Applied Biosystems) was used for calculation of the cycle threshold (CT) values and quantification of the number of H. pylori cells. The number of cells and the standard deviations are means of independently performed duplicate experiments.