10cm flasks

Plp1::GFP mice aged 12-16 weeks were euthanized and their small intestine was removed from duodenum to terminal ileum. The longitudinal muscle-myenteric plexus (LMMP) layer, which contains myenteric ganglia, was carefully dissected from underlying tissue under a dissecting microscope in ice-cold PBS supplemented with 10% bovine serum albumin. After dissection, LMMP tissue was digested for 60 minutes at 37° C in dispase (250 μg/ml; STEMCELL Technologies, Vancouver, BC) and collagenase XI (1mg/ml; Sigma-Aldrich, St. Louis, MO). Following digestion, the cells were filtered via a 40 micron filter to ensure a single-cell suspension. Immediately after digestion and filtering, cells were counted and resuspended at a density of 10⁵ cells/mL in a 1:1 mixture of DMEM (Thermo Fisher, Waltham, MA) and NeuroCult Basal Media (STEMCELL Technologies, Vancouver, BC) supplemented with 20 ng/mL FGF, 20 ng/mL IGF1, 2% B27 supplement, 1% N2 supplement, 50 mM b-mercaptoethanol, and 75 ng/mL retinoic acid. 10⁵ cells/mL in a total volume of 10 mL were placed 10cm flasks (Corning Inc, Corning, NY) at 37° C and 5% CO₂ for 7-10 days. Media was replaced on day 5 by centrifuging cells at 250g for 3 minutes followed by re-suspension in fresh media and return to the same flasks and incubator for 5 more days. Cells from both male and female mice were used and were cultured separately.