Relative mRNA accumulations in cells harvested at 24 h were measured by two-step reverse transcription quantitative real-time PCR (Q-PCR) using SYBR Green chemistry. Total RNA was extracted from cells stored in RNAlaterH using an Ambion RNAqueousH-Micro kit (AM1931) and converted to cDNA using an Applied Biosystems High capacity RNA-to-cDNA kit (4387406). All samples were DNase-treated and periodic controls lacking a reverse transcriptase enzymatic step indicated that DNA contamination was negligible. Real-time PCR primers for the cytokines, transcription factor and endogenous control genes were designed from conserved regions identified in multiple alignments of rodent sequences in the NCBI database. For TLR genes de novo amplification and sequencing of a larger (300-500 bp) fragment was first carried out using conserved primers (designed as above). Real-time PCR primers were then designed from the resulting Apodemus sylvaticus TLR sequence. All primers were designed with Primer 3 v. 0.4.0. For each target and endogenous control gene 2-6 primer pairs were designed and trialled and the optimal set selected for further use. All selected primers (Table 1) were confirmed to be specific by re-sequencing of the target fragment, did not produce confounding primer dimers under assay conditions and had approximately equal efficiencies close to 100%, based on the average of 5-fold dilution series repeated on multiple different plates. Target genes were normalized against the endogenous control gene b-actin. This was the most stable endogenous control gene amongst a set of 9 candidates considered (including c-actin, G6PDH, GAPDH, HPRT1, Tubulin, YWHAZ, 18 s and Albumin) as determined by stepwise exclusion of those with low stability estimated by the M statistic [27] . This procedure was conducted separately for 10 LPS-, HKLM-or ODN 2006-stimulated cultures randomly selected out of a pool of 300 culture samples. In all three cases b-actin was identified as the joint most stably expressed gene. The primers used for b-actin were sense 59-AGATCAAGATCATTGCTCCTCCT -39 and antisense 59-TCCTGCTTGCTGATCCACATC -39 which gave a 102 bp amplicon. The 96-well real-time PCR assay was designed so that all target genes for one animal were run on a single plate, except for Foxp3 amplifications which were all run on a separate plate. Each target gene reaction and the endogenous control gene reaction were run in duplicate for samples and in triplicate for a reference cDNA that was included on each plate. A no-templatecontrol was included in duplicate for each primer pair. Real-time PCR assays were run using default settings and the manufacturerrecommended enzyme, buffer and SYBR Green reagents on an ABI 7500 Fast machine (Applied Biosystems). ABI 7500 v 2.0.4 software was used to calculate the expression of each target in each sample as a fold difference relative to the reference cDNA by the DDCt method [28] .