Hello everyone!

I am currently trying to improve my protocol for isolating dopaminergic neurons. Briefly, I dissect ventral midbrain from E14.5 mice (aprox. 10 embryos) and dissociate cells with a papain digestion and mechanically. My culture medium only consists in DMEM with horse serum, fetal bovine serum and gentamicin, with medium changes twice a week (removing FBS and adding AraC for avoiding glial proliferation), but I get a low cellular yield (sometimes lower than 10^6cells/mL). I seed them in precoated (PDL/PLL + laminin) 24well plates, with 100.000cells/well. Is there any way to improve this yield and survival, apart from avoiding overtrituration and reducing dissection time? And is it possible to isolate only dopaminergic neurons or increase its number in the culture (selective medium, neurotrophins, gradient...)?

Also, after 4 days in culture I see this brilliant points increasing (cell death) and a lot of variability depending on the density (although I try to really seed 100.000 cells in every well).

Thank you!!