Dissociated neuronal cultures and Immunocytochemistry

After procurement, DRGs (donors 91 and 92, Supplemental Table 1) in aCSF were transported to the lab over ice (~30 minutes). The DRGs were trimmed of excess connective tissue, fat, and nerve roots to expose the DRG bulb. The DRG bulb was then cut into 3mm sections and placed in 5mL of pre-warmed digestion enzyme containing 1 mg/mL of Stemzyme I (Worthington Biochemical, LS004106), 0.1 mg/mL of DNAse I (Worthington Biochemical, LS002139), and 10ng/mL of recombinant human β-NGF (R&D Systems, 256-GF) in HBSS without calcium and magnesium (Thermo Scientific, 14170-112). The tubes were placed in a 37° C shaking water bath until the DRG sections dissociated (4–10 hours). The solution was then filtered through a 100μm mesh strainer. The resultant cell suspension was then gently added to a 15mL tube containing 3mL of 10% Bovine Serum Albumin (Biopharm, 71-040) in HBSS. The tubes were then centrifuged at 900g for 5 min at room temperature. The supernatant was aspirated, and the pellet was resuspended in prewarmed BrainPhys^® media (Stemcell technologies, 05790) containing 1% penicillin/streptomycin (Thermo Fisher Scientific, 15070063), 1% Glutamax (Thermo Scientific, 35050061), 2% NeuroCult SM1 (Stemcell technologies, 05711), 2% HyClone^™ Fetal Bovine Serum (Thermo Fisher Scientific, SH3008803IR), 1% N-2 (Thermo Scientific, 17502048), 0.1% 5-Fluoro-2′-deoxyuridine (FRDU, Sigma-Aldrich, F0503), and 10ng/ml of β-NGF. Cells were plated in a 24 well plate containing 12mm coverslips which were pre-coated with 0.1mg/mL of poly-D-lysine at a seeding density of 100 neurons per well. Cells were incubated at 37°C and 5% CO2 for 3 hours to allow for adherence. Following neuron adherence, wells were flooded with 1mL of prewarmed media, and half media changes were performed every other day.

On DIV 3 (donor 91) and DIV 5 (donor 92), cells were washed with 1X PBS and fixed with 10% formalin for 10 min at room temperature. Cells were then washed 3 times with 1X PBS and blocked with 10% normal goat serum in PBS for 1 hour at room temperature. Cells were then permeabilized with 10% Normal Goat Serum and 0.3% Triton X in PBS for 30 min at room temperature. To label neurons, cells were incubated with peripherin (1:1000, Supplemental Table 5) diluted in blocking buffer overnight at 4°C. The next day cells were washed 3 times with 1X PBS and incubated with a goat anti-chicken 488 secondary antibody (1:2000) and DAPI (1:5000, Cayman Chemical, 14285) diluted in blocking buffer for 1 hour at room temperature. Cells were washed with 1X PBS and then covered with True Black (diluted at 1:20 in 70% Ethanol; Biotium; 23014), a blocker of lipofuscin, for 1 minute. The cells were washed again in 1X PBS. Coverslips were lifted out of the 24 well plate and mounted onto glass slides with Prolong Gold Antifade reagent (Fisher Scientific, P36930). A negative control coverslip was processed similarly in each experiment but was not exposed to the primary antibody. All images were taken on an Olympus FV3000 confocal microscope at the University of Texas at Dallas.