Type 3

Mice were killed and the spinal column was quickly dissected out with sympathetic chain and spinal roots attached. Figure 1A provides a simplified schematic of the anatomic organization of intraspinal preganglionic and paravertebral postganglionic neurons. The remaining tissue was incubated in continually oxygenated ACSF containing collagenase (20-mg Type III per 1-ml ACSF, Worthington Biochemical Corporation) for 1.5 h. ACSF used for incubation was buffered with either bicarbonate or HEPES. No difference was observed as a result of incubation buffer. Following incubation, tissue was vortexed to remove adherent fat and washed with ACSF several times to eliminate residual collagenase. The intact sympathetic chain was removed by severing rami, and was then pinned down into a clear Sylgaard recording dish (Fig. 1B), through which recirculating, oxygenated ACSF was continually perfused.

For ear preparation, dorsal and ventral layers of the ear were separated and incubated in RPMI (Gibco) with 250 μg/mL Liberase TL (Roche) for 90 minutes at 37°C in 5% CO₂. Skin was then dissociated using a 40 μm cell strainer (BD Pharmingen) and resuspended in complete RPMI media (cRPMI) containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 55 μM 2-Mercaptoethanol. For flank skin preparation, a section of skin was harvested from the flank following hair removal with an electric trimmer equipped with a two-hole precision blade (Wahl). Skin sections were then minced with a sterile scalpel blade into ~2mm sections, and incubated in RPMI containing 1 mg each of type III and type IV collagenase (Worthington) for 120 minutes with vortexing every 30 minutes. The resulting solution was passed through a 40 μm cell strainer and resuspended in cRPMI. Bone marrow derived dendritic cells for restimulations were generated by culturing C57BL/6 bone marrow in GM-CSF supplemented cRPMI for 7–11 days. BMDCs were then harvested and infected 5–8 hours with stationary phase L. major at a ratio of 10:1 in the presence of 1 μg/ml CpG and LPS. Infected BMDCs were incubated at a ratio of 1:5 with 10⁶ skin cells in 24 well plates for 12–16 hours. Cells were incubated for the last 4 hours with 5 μg/ml BFA (eBioscience), stained for IFNγ, and analyzed by flow cytometry.