Isolation and culture of mouse myenteric neurons

Myenteric neurons were dissociated from mouse small intestine, using a previously described method19 (link)39 (link). Cell cultures were prepared by adding 50 μL of a constantly mixed cell suspension into 8-well chambers (30108, SPL, Saveen Werner, SE) prefilled with 450 μL of Neurobasal A (NBA) cell culture medium (NBA, Thermo Fisher Scientific, SE with 10%v/v fetal bovine serum FBS, BioWest, FR, 0.5 mM L-glutamine K0282 and 50 U/mL penicillin, 50 μg/mL streptomycin A2213, BioChrom AG). From each animal two 8-well chambers (69 mm²/well) were prepared. Fresh medium containing applicable experimental test agents was added to cultures after a 4 day equilibration culture period. Control wells were cultured in parallel. Cells were fixed in Stefanini’s fixative, rinsed in Tyrode solution, frozen, thawed and subjected to immunocytochemistry.