Macrophage Gene Expression Analysis

Thioglycollate-elicited macrophages were harvested by peritoneal lavage in sterile saline and pooled from 2–4 mice per strain per experiment as previously described^{(26 (link))}. Cells were stimulated with E6020 at concentrations ranging from 0.1 ng/ml – 100 ng/ml for 2 hours after which macrophages were harvested in TriPure reagent (Roche, San Francisco, CA) and frozen at −80° C. RNA was extracted per the manufacturer’s protocol and cDNA was reverse transcribed from 1 μg RNA per sample using Qscript kits (QuantaBio, Beverly, MA)^{(26 (link))}. qRT-PCR was performed on an ABI 7900HT instrument (Applied Biosystems, Waltham, MA) using Power SYBR Green PCR master mix (Applied Biosystems) with primers (Sigma-Aldridge, Inc., St. Louis, MO) for Hprt, Tnf, Il1b, Ifnb1, and Cxcl10^{(27 (link))}. Levels of mRNA for specific genes are reported and statistical analyses performed on negative delta cycle threshold (C_T) values, as normal distributions are preserved in this method. Data are displayed as arithmetic mean ± SEM. Conversion to relative gene expression normalized to that of WT unstimulated macrophages (“fold induction”)^{(28 (link))} are also shown on the right y-axis (Figure 1). The housekeeping gene encoding hypoxanthine-guanine phosphoribosyltransferase (Hprt) was used for normalization of RNA levels within each sample.