Earle s balanced salt solution

hCO and hSO were dissociated as previously described^{57 (link)} with some modifications. Briefly, organoids (single organoids cultured in a 96-well plate or up to eight organoids cultured in a six-well plate) were incubated with an enzyme solution containing 10 U ml⁻¹ papain (Worthington, catalogue no. LS003127), 1× Earle’s balanced salt solution (Sigma-Aldrich, catalogue no. E7510-500ML), 0.36% d(+)-glucose (Sigma-Aldrich), 26 mM NaHCO₃ (Sigma-Aldrich), 0.5 mM EDTA (Sigma-Aldrich), DNase (Worthington, catalogue no. LS002007), 6.1 mM l-cysteine (Sigma-Aldrich) and the ROCK inhibitor Y27632 (10 µM) at 37 °C with 5% CO₂ for 15 min; this solution was prewarmed at 37 °C for 30 min to activate papain, and then cells were washed with a prewarmed protease inhibitor solution (1× Earle′s balanced salt solution, 0.36% d(+)-glucose, 26 mM NaHCO₃, 0.2% trypsin inhibitor (Worthington, catalogue no. LS003086)). Organoids were then triturated, and the resulting single-cell suspension was centrifuged at 1,200 rpm for 5 min. The pellet was resuspended with 3% BSA (Sigma-Aldrich) and then filtered.

Dissected ipsilateral or contralateral retinas were digested in papain solution (20 U/mL in Earle’s balanced salt solution, Worthington, Freehold, NJ) followed by repeated trituration in DMEM/F12 + 10% fetal bovine serum + DNaseI. Dissociated cells were washed with PBS, resuspended in PBS + 2% fetal bovine serum, passed through a 0.45-µm cell strainer, and kept on ice before and throughout flow cytometry. Rhodamine-positive neurons, with exclusion of DAPI-stained dead cells, were collected using a BD FACSAria cell sorter in the Columbia University Cancer Research Center core facility.

Sympathetic neurons were prepared in a manner previously described [13 (link)]. DBA/J mice at 2 to 3 months of age were sacrificed by cervical dislocation. The superior and stellate ganglia were removed under stereomicroscopy and immediately placed in chilled (4–8°C) sympathetic complete medium [DME-F12 medium supplemented with sodium bicarbonate, HEPES, penicillin/streptomycin, 5% fetal bovine serum, and MITO⁺ Serum extender (Collaborative Res, Bedford, MA, USA)]. The ganglia were then incubated for 20 min at 37°C in a Earle’s Balanced Salt solution (Sigma, St. Louis, MO, USA) containing 10 U/ml papain (Worthington Biochemical Corp., Lakewood, NJ, USA), followed by a 30-min incubation in Earle’s Balanced Salt solution supplemented with 1.3 mg/ml type II collagenase (Worthington) and 2.2 mg/ml dispase II (Roche, Indianapolis, IN, USA). Cells were then dissociated by trituration with a fire-polished glass Pasteur pipette in trituration solution (sympathetic complete medium containing 1.8 mg/ml bovine serum albumin) and plated on poly-D-lysine–coated No.1 circular glass cover slips. The isolated neurons were maintained in 5% CO₂-95% O₂ at 37°C in trituration solution and used ~24 hours after plating. All procedures were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee.