Cell viability Cell viability was determined using alamarBlue (In

Cell viability Cell viability was determined using alamarBlue (Invitrogen). Briefly, cells were seeded in a 96 well plate at 2×105/ml. After 6 hours of cell adherence, cells were treated in the presence and absence of RBE for 24 hours at 37°C, 5% CO2 in maintenance media. Supernatant was removed and alamarBlue was added to media (20 μg/ml). Fluorescence was detected at excitation:

530/25; emission: 590/35 in ELISA plate reader (Bio-Tek Synergy HT, Winooski, VT). Bacterial quantitation RBE doses of 0, 1, 2, 5 and 10 mg/ml were tested for direct effects on Salmonella viability. Bacteria was added to media at a concentration of 2 × 107 CFU/ml and incubated for 6 hours at 37°C. Bacterial suspension was serially diluted, plated on agar plates and counted after 24 hours incubation. Quantitative PCR for Lactobacillus spp DNA was extracted from fecal pellets of control and rice bran fed mice before and Selleckchem SP600125 after Salmonella challenge using a MoBio Powersoil DNA extraction kit (MoBio, Carlsbad, CA). A dilution of DNA from pure cultures of Lactobacillus rhamnosus was used to generate standard curves and DNA from Pseudomonas aeruginosa were run as a negative control to ensure primer specificity. DNA was quantified by Nanodrop (Thermo Fisher Scientific) and diluted to 5 ng/μl. Real time PCR primers were used from Malinen et al. [47] for amplification of Lactobacillus spp. Samples were run on an ABI Prism 310 thermocycler (Applied Biosystems)

using the following program: 95°C for 3 min 30 s followed by 30 cycles of 95°C for 15 s, 58°C for 20 s 72°C for 30 s and melt curves PND-1186 molecular weight were generated by 95°C for 1 min followed by eighty 10 s repeats at set point temperatures incrementally decreasing by 0.5°C. Statistical analysis Data was analyzed using Graphpad Prism5 Software. Experiments

were repeated a minimum of three times. Carnitine palmitoyltransferase II Raw data were log transformed into a log10 scale for CFU analysis and repeated measures ANOVA and post hoc Tukey’s test were used for Salmonella fecal shedding and fecal Lactobacilli measures. Inflammatory cytokines were analyzed using two -way ANOVA and Silmitasertib order Bonferroni post hoc test. A nonparametric ANOVA (Kruskal Wallis) was performed, followed by Dunn’s test for in vitro Salmonella assays. Significance was determined for all studies at P <0.05. Acknowledgements We would like to thank Dr. Andres Vazquez-Torres for providing the strain of Salmonella used in these studies, and Dr. Anna McClung from the USDA-ARS Dale Bumpers Rice Research Center for providing rice bran from the single Neptune variety. We also thank Dr. Daniel Manter from USDA-ARS-Soil Plant Nutrient Research, Brittany Barnett for for assistance with qPCR of Lactobacillus spp. and Adam Heuberger and Caleb Schmidt for their technical assistance. Funding A Grand Explorations in Global Health Grant from the Bill and Melinda Gates Foundation (OPP1015267) and the Shipley Foundation supported this work.

Comments are closed.