Quantifying DRG Neurite Length with Phentolamine

Based on the protocol described previously^{8 (link)}. 4–6-week-old C57BL/6J mice dissociated DRG neurons were plated onto the poly-D-lysine (100 µg/mL), laminin (10 µg/mL) and the aggrecan (50 µg/mL) (P-6407, L-2020, A-1960, Sigma) coated coverslips in the 24-well culture plates and cultured in Neurobasal medium (1088802; Thermo Fisher Scientific) with B27 supplement containing penicillin, streptomycin, 1 mM l-glutamine, 50 ng/mL NGF, 2 ng/mL GDNF, and 10 mM AraC at 37°. For drug treatment, DRG neurons were cultured for 72 h in the presence of the drug phentolamine at 1, 3, 5, 8, 10, 12, 20 μm concentrations. The cells were post-fixed 4% paraformaldehyde (PFA) followed by phosphate buffer saline (PBS) washing and immunostained with anti-mouse b-III-tubulin (1:1000; 801201, BioLegend). Coverslips were then inverted and mounted on the glass-slides using Prolong Gold antifade reagent with DAPI (P36935, Fisher Scientific). Images were captured covering the entire coverslip area at × 20 magnification using an Olympus Fluorescent Microscope. Total neurite length was quantified with an ImageJ plug-in, NeurphologyJ^{38 (link)} (RRID: SCR_003070). Neurphology J operates on the entire image and quantifies the neurite length in pixels. Data were obtained from at least 4 separate experiments repeated in duplicates.