To click here assess whether Inhibitors,Modulators,Libraries these substances have potential to modulate a Salmonella induced response in enterocytes, we tested their effect on a Salmonella induced IL8 and NFKBIA mRNA response in IPEC J2 cells. Methods Small intestinal segment perfusion with Salmonella Total RNA isolated from mucosal scrapings of an earlier described SISP experiment was used for micro array and QRT PCR analysis. In the mid jejunum, intestinal seg ments were prepared in 4 male piglets as described. Inhibitors,Modulators,Libraries 10 cm of the control seg ment was dissected before segments were perfused for 1 hour without and with Salmonella enterica subspecies enterica serovar Typhimurium DT104 109 CFUml according to the scheme depicted in Figure 1A. Subsequently, loops were perfused for 1, 3 or 7 hours without Salmonella and samples were dis sected at 2, 4 and 8 hours after the first exposure with Salmonella.

Details of this SISP experi ment, dissection of mucosal scrapings, and RNA iso lation from these scrapings was published previous. RNA from mucosal scrapings Inhibitors,Modulators,Libraries of three of these four pigs was stored as alcohol precipitate at 20 C. After centrifugation the RNA pellet was dissolved in RNAse free water, and the integrity of this RNA was checked by analyzing 0. 5 ug on a 1% agarose gel. The SISP experiment described in Niewold et. al. was approved by the Animal Ethics Commission in Lelystad, the Netherlands, in accordance with the Dutch law on animal experimentation. Microarray analysis The commercially printed Inhibitors,Modulators,Libraries Pig Operon expression micro array was used for all hybridizations.

Inhibitors,Modulators,Libraries Array slides contained a total of 13297 70 mer oligonucleotide se quences representing 10655 Sus scrofa sequences with a blastn hit to known human, mouse or pig mRNA se quences and some 3 expressed sequence tags. All probes were printed in duplicate. Dual labeling of total RNA using the RNA MICROMAX TSA labeling and detection kit, hybridization and washing of slides www.selleckchem.com/products/CAL-101.html was performed as described recently, except that 4 ug of template was used instead of 1 ug. A total of 6 comparative hybridizations were performed according to the scheme depicted in Figure 1B and C. For each comparison a dye swap was performed. Slides were scanned and images were gridded on a GenePix 4200A 01 Autoloader 116826. Data files were processed in GenePix Pro 6. 1. 0. 4 or 6. 0. 1. 25. Data normalization was performed using a customized version of the statistical software package R for simultaneous data analysis of dye swaps. Significantly differential expressed probes with M value of 1. 58 or 1. 58 and with a p value 0. 025 were selected. For each probe 4 spots were hybridized, 2 on one slide and 2 on the dye swap slide. Probes with more than one missing values were removed from gene lists used for bioinformatics analysis.