Cold Challenge Experiments on Mice

Male mice, 8-12 weeks old, were used in all experiments. Breeding pairs of wild type and STAT6^−/− mice on BALB/cJ background were purchased from the Jackson Laboratory, and IL4/IL13^−/−, IL4Rα^L/L, and LysM^Cre mice on the BALB/cJ background were obtained from the Locksley or Brombacher laboratories. For cold challenge experiments, mice were fed ad lib and individually housed in cages that had been pre-chilled at 4 °C^{8 (link)}. Core body temperature was monitored hourly by a rectal temperature probe (Physitemp). For the thermoneutrality experiments, mice were adapted to 30 °C in laboratory incubator (Darwin Chambers) for 2-4 weeks prior to experimentation. For rescue experiments, the β3-adrenergic agonist CL-316243 (Sigma) was injected intraperitoneally at 0.1mgkg⁻¹ 30 min before the cold challenge. Tissues were harvested at the end of 6 h cold challenge, and processed for RNA and protein analyses. To deplete macrophages, mice were injected intraperitoneally with two doses of clodronate-containing or empty liposomes (400 μl and 100 μl at 24 h and 30 min, respectively, prior to initiation of experiment)^{22 (link)}. Depletion was confirmed by flow cytometric analysis of monocytes and macrophages in blood, adipose tissues, and spleen. Cohorts of ≥ 4 mice per genotype or treatment were assembled for all in vivo studies, which were repeated 2-3 independent times. All data are presented as mean ± s.e.m.