Hi everyone,
I would like to design an apoptosis induction experiment on mouse primary cortical and hippocampal neuron culture. The annexin V/PI kit seems to be the best for this. I would like to mesure in 96 well plate, but first I have to detaching the cells. The tripsin treatment seems too harmful and I am worry about the cell death, that the 90% of my culutre will be annexinV positive.
Thanks!
Dear Réka,
normally for flow cytometry protocols we use gentle dissociation methods, such as by using Accutase(R) or even EDTA. If your are going to use EDTA, try a very diluted concentration, such as 500 μM. You can add 90 μL/cm² and agitate it for 20 min, then collect the cells.
Hope it helps,
Eduardo.
Although I have not tried this, I read that if you plate on collagen, use collagenase as dissociation. I have cultured primary neurons on collagen and know that they do quite well on. We have tried to dissociate DIV10 primary neurons using both trypsin and accutase for FACS and ended up with a 30% cell death. Maybe we were just not gentle enough?
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