DRG culture and neurite outgrowth assay

The cultures of DRG neurons were prepared as described by Paveliev et al.³⁶ with small modifications. DRGs were dissected from the spine of the healthy male P30 Wistar rats purchased from The Laboratory Animal Centre of the University of Helsinki. The permission to use experimental animals for research purposes (KEK14-019) was given by The Laboratory Animal Centre of the University of Helsinki. Ganglia were cleaned from nerve fibers in PBS and washed two times with Ca²⁺ and Mg²⁺ free HBSS (ThermoFisher Scientific). The ganglia were incubated in HBSS containing trypsin (5 mg/ml) and collagenase/dispase (10 mg/ml) for 30 to 60 min at 37°C, washed once with HBSS-DNase I solution, and triturated in HBSS-DNAase I solution until disappearance of visible tissue pieces. Supernatants were transferred to the fresh tube, the cells were precipitated by centrifugation at 110 g for 5 min, washed two times with neuron culturing media (1xNeurobasal medium, 1xB-27, 100 µg/ml primocin, 25 μM L-glutamine, 0.1% DMSO) and plated on poly-ornithine and laminin-precoated coverslips. Neurons were cultured in the presence of 0.5–10 µM BT44 or ARTN (30 ng/ml) for a positive control for 16–20 h. Afterwards, the DRG neurons were fixed with 4% PFA for 10 min at RT, washed with PBS, and permeabilized with 1xPBS containing 0.1% Triton X-100 (PBS-T). DRG neurons were probed with 1:500 rabbit anti-PGP9.5 antibodies (Enzo Biochem Inc., USA) and visualized with anti-rabbit secondary antibodies conjugated with Alexa488 1:200 (Invitrogen). Mounting was done with Imu-mount (Thermo Scientific). The number of PGP9.5-positive neurons with neurites at least twice as long as the body was quantified under fluorescent microscope (Zeiss5) and normalized to the total number of neurons. The counting in random vision fields on coverslips was carried out by the observer who was blinded to the treatment groups. Experiments were performed at least in triplicates and each concentration of BT44 was evaluated in four to five independent experiments. At least 70 neurons per treatment group were analyzed in each experiment. For the statistical analysis, the data for technical replicates were averaged. For representation purposes, the data were converted to percentages from the values in vehicle-treated wells for each experiment and the results for all concentrations were pooled together.