Primary Hippocampal cell culture-DEAD! - (Sep/28/2006 )

Hi All!!

I have just started with primary hippocampal cell culture. I had isolated the embryos and dissected the hippocampus in CMF HBSS. The cells were plated in Neurobasal supplemented with B27,glutamine,superoxide dismutase,glutathione,antibiotics(PSN). The culturel medium was switched to cytosine arabinoside containing medium on the fourth day and maintained so till the eighth day.The cells were fine till then. Then as per the protocol they were switched back to the normal (cytosine free medium). And two days later(tenth day of culture) I notice that allmost all the cells are dead!!! Can anyone help me? Is it the pH? Or some contamination I cannot see? I have heard of mycoplasma affecting cultures...No idea how that looks...Iam scared of doing the next batch of culture only to see them all dead in ten days.

usually changing media for hippocampal neurons will kill them as they dont tolerate it. we never changed media for hippocampal after they r plated. if u want to change or add then remove half the media and replace the other half.

Hi,

Instead of giving 100% change of medium, could you change medium gradually. Like 25% new medium + 75% old medium, then 50% + 50% and then 75% new + 25% old and finally 100% new medium.

Hope it helps.

Pravin

Thanks for the answers....I did change medium as 75% new, 25% old...and did it as fast and gentle as I could....Will try the 75% old, 25% new. Thnks again..and anymore suggestions is always welcome!