I agree with Abdelaziz. The second parameter to take into consideration is the medium used to dissociate the tissues. I recommend the use of Leibovtiz L15 medium which allow to work at constant pH (since this medium does not contain sodium bicarbonate).

Hi Claudia,

I've been doing this type of culture from mice (genotyping before culturing) almost weekly for some years. After taking the tails for genotyping, normally I dissect the brain in room temperature HBSS, separating the hemispheres and removing the meninges. Then I keep the hemispheres in HBSS in the fridge until PCR is almost done so I have time to dissect cortices and hippocampi. Then I follow with the trypsination. I try to calculate the time so By the time I'm in the trypsin incubation the PCR results are finished so I would only proceed with the dissociation of those tissues of interest.

Hope it helps.

Hi Claudia,

you might want to try preserving the tissue in Hibernate-E medium from ThermoFisher, once complemented with B-27 it will allow you to store it for long periods in ambient CO2 levels. Check their product page for more information. It usually works quite well for me. 

Good luck!

I've found that allowing the tissue to sit (under any conditions) while genotyping severely lowers neuron viability, especially for long-term cultures using serum free conditions. 

I usually culture and plate cells from all embryos or have someone else help with the genotyping while the culture is being performed. Tissue samples are collected prior to starting cortical dissections. Genotyping is done in less than an hour using Sigma Extract-N-Amp kit and the results are ready before the culture is finished. 

Good Luck!