Dy479 05

DRGs from miR-21–cKO mice and their littermate controls were collected and placed into Ham’s F-12 Nutrient Mixture (Gibco). DRGs were then dissociated using 3 mg/mL Dispase (Roche), 0.1% collagenase (Sigma-Aldrich), and 200 U/mL DNase I (Sigma-Aldrich) in F-12 medium (Gibco). Thereafter, DRGs were triturated, and cell suspensions centrifuged at 300g for 6 minutes. Pellets were resuspended in fresh DRG medium and plated on glass coverslips precoated with poly-L-ornithine (100 μg/mL; Sigma-Aldrich) and laminin (40 μg/mL; Roche). Cultures (10,000 cells/well) were incubated at 37°C for 24 hours, and then stimulated with vehicle or capsaicin (1 μM) for 3 hours. Culture media were removed, and CCL2 was measured using ELISA (R&D Systems, DY479-05) in DRG neuronal culture media, according to the manufacturer’s instructions.
Transmigration assay was performed using cell culture inserts (Transwell plate, Costar) with 8-μm porous polycarbonate filters. DRG neurons (10,000) were cultured in 300 μL F-12/10% FBS in the lower compartment for 24 hours, and then 40,000 WT BMDMs treated with vehicle or CCR2 antagonist (1 μM; Merck) were added to the upper filter. DRG neurons were then stimulated with capsaicin (1 μM) for 3 hours.