Thank you so much for your response. I used to isolate from E16 embryos. But currently I am trying to work with P(0-2) pups, the same protocol doesn't work as well. All my buffers and media are Ca2+/Mg2+ free. I use 20U papain with DNAse (30 minutes incubation at 37deg). I came across that the postnatal isolations include more debris. So I included another step; layering the triturate (after passing through 70 µm filter) over 1% BSA. I plate the cells with NB+B27+L-Gln. I change 50% media every 3 days.

Thank you for your protocol. I tried to include serum in my plating media and changed it after 1 hour. But it increased the glial population. Do you treat the cultures with Ara-C, does that affect the cell viability.

THank you so much for your response Edgar! I will definitely try reducing the incubation time and maybe without the 1%BSA. I haven't been able to maintain the cultures post 10days to reach 5 weeks of culture. I will also keep an eye about the evaporation.

Srinidhi

I was using this protocol - https://www.nature.com/articles/nprot.2012.099.pdf - and almost never had issues with yield and survival.

I did not 'bake' coverslips (just leave them in HNO3 overnight, then wash with plenty of water). Even without HNO3 treatment the neurons attach to the glass well enough but tend to form tight bundles later.

I tried papain as well and did not notice any advantages over trypsin.

Thank you Misha. I did try this protocol this time. I somehow found the neurons to cluster. I had this issue in the beginning stages. I changed from PLL to PDL and used a fresh dilution of PDL (50µg/mL). They seem to be fine on the dish but don't like the coverslips. I used HCL instead of nitric acid, baked at 50deg for 3 hours before rinsing with tap water and thrice with ddH2O. I coated with PDL for 1hr at RT before washing thrice with ddH2O and air drying. I find more death on the coverslips, even if I seed 600K in 35mm dishes.