k mechanism to promote Notch activated transcription and the network analysis suggests that this interaction may be mediated by a direct contact with Notch itself. This study enhances knowledge on the chicken macrophage transcriptional response to endotoxin by elucidating the complex gene networks involved in the chicken inflammatory response and reports the novel involvement of NLRC5. Methods Cell Culture and Stimulation The chicken HD11 macrophage cell line was cul tured in RPMI 1640 medium supplemented with 10% heat inactivated newborn calf serum, 2 mM gluta mine, 1 mM sodium pyruvate, 0. 1 mM non essential amino acids, 100 U ml penicillin, 100 ug ml streptomy cin, 10 mM HEPES and 5 �� 10 5 M 2 mercaptoethanol at 41 C and 5% CO2. Cells were plated in 75 cm2 tissue flasks and cul tures were split every 3 days.

Cell viability was 90% by trypan blue exclusion. Prior to sti mulation with endotoxin dissolved in Phosphate Buffer Saline, cells were cultured at an initial density of 2. 8 x106 cells flask into 25 cm2 tissue flasks and kept overnight in the incubator, then stimulated with 0. 0, 0. 1, Drug_discovery 1. 0, 10. 0 ug ml endotoxin which was isolated from Salmonella typhimurium 798 utilizing the aqueous buta nol 1 extraction procedure as described by Morrison and Leive 1975. Cells were collected at 1, 2, 4, and 8 hours after endotoxin stimulation. RNA Isolation, DNase Treatment and QPCR Experiments Total RNA was isolated from pooled samples using RNAquous? accord ing to manufacturers instructions. The mRNA expres sion levels of TLR15, IL1B, IL6, IL10, IL8, and IFNG were determined by quantitative real time RTPCR, using QuantiTect SYBR Green RT PCR.

Each RT PCR reaction was run in triplicate for each sample and consisted of either 50 ng or 75 ng total RNA, 12. 5 ml QuantiTect SYBR Green master mix, 0. 25 ml QuantiTect RT mix, forward and reverse primers, and RNAse free water for a final volume of 25 ml. The QPCR primer sequences have been previously published. The QPCR reactions were performed on an Opticon 2. An initial 50 C step for 30 min was followed by 95 C for 15 min and 40 cycles for all PCR amplifications. Gene slopes were determined with serial dilutions differing by 10 fold. A melting curve from 60 to 90 C with a reading at every 1 C was also performed for each individual RT PCR plate. Adjusted cycle threshold values were calculated as follows, 40 for all genes except IFNG.

The threshold of 40 cycles was raised to 45 cycles for IFNG, because most adjusted cycle numbers were greater than 40. Mean adjusted C values of each triplicate of assays were used in statistical analysis. All RNA samples were DNase treated with DNA Free according to manufacturers instructions before QPCR. The fold changes in mRNA levels were determined as follows, C non stimulated C target gene non stimu lated C 28 s non stimulated. C stimulated C target gene stimulated C 28 s stimulated. The fold change in mRNA 2 non stimulated C stimulated Statistical Analysis of QPCR Data The