Recombinant mouse ccl2

To assess the outgrowth of peripheral neurons in response to injury, we evaluated neurite outgrowth in explanted ganglia (Shoemaker et al., 2005 (link)). Three weeks after intrathecal injection of the virus, L5 DRGs from uninjured CCR2 −/− and WT mice were removed, desheathed, placed on coverslips, and overlaid with 7.5 μl Matrigel (Becton Dickinson, Franklin Lakes, NJ). Culture plates were placed in an incubator at 37°C for 5 min to allow gelling of the Matrigel before adding 1 ml F12 medium with the additives described in Hyatt Sachs et al. (2010) (link). Phase-contrast images of neurite outgrowth from each DRG were captured at 24 and 48 h after explantation using an Axiovert 405 M microscope at 10x magnification. Neurite outgrowth was assessed using MetaMorph software by measuring the distance between the edge of the ganglion and the leading tip of the longest 20 processes in each explant. The average length of the 20 longest neurites is taken for each ganglion. At 48 h, explants were fixed and labeled with an antibody against βIII tubulin (1:500; Promega) to visualize the outgrowth. Representative images were taken at 10×. In some experiments, 200 ng/ml of recombinant mouse CCL2 (R&D Systems; Minneapolis, MN) was added to the culture medium at the time of plating.