primary cell culture death - (Aug/27/2006 )

As my PhD thesis I start the fetal hippocampal cell culture. But I have a great problem and it is that my cells -nearly all- are died after 24 h. At first I thought it was because of my powder medium and I changed it to solution medium, but it was not effective. I have sterilized my incubator several times-160 c for 3 hour- but again there is not any progress in my work.
My media is DMEM-high glucose+ FBS 10% . can anyone help me?

We use Neurobasal medium for primary hippocampal neurons, actually for all our neuronal cultures we use NeuroBasal media supplemented with B-27.

One has to coat plates to have the cells attached and b gentle thru out the whole procedure.

Good Luck !!!

Dear Scolix, Thanks for your reply. Do you think that the reason of my cell death is my media? As I know many labs use this media?

Hi maryam,

One cannot b 100% sure of the reason as primary cultures r very tricky and every reagent that u r using could b the reason.

Using FBS, one is promoting non neuronal growth compared to neuronal cultures and this may interfere with neuronal growth.

Hi Scolix. I changed the media to neurobasal + B27 but the problem is still present. What can be the cause? I use PBS for trypsinization of the tissue. can pbs kill my cells.
And I like to know for the detecting cell death in the cell culture dishes how much be the concentration of trypan blue and how much time must be allowed to cell to dye?
Thanks

Scolix gave another good hint regarding coating; I am not familiar with your special type of cells but precoating of plates or wells for primary culture is a MUST; may be you find good literature or try several different commonly used; mixtures as matrigel from BD may help if nothing else works

Hi Scolix. I changed the media to neurobasal + B27 but the problem is still present. What can be the cause? I use PBS for trypsinization of the tissue. can pbs kill my cells.
And I like to know for the detecting cell death in the cell culture dishes how much be the concentration of trypan blue and how much time must be allowed to cell to dye?
Thanks

Hi Maryam
I haven't work with these cells before but have a lot of experience establshing primary cultures of various sorts.
Just as a long shot, is your media being buffered correctly?

Hi again Maryam
Do you know anyone who has tissue culture experience that can help you? It sounds to me like your problem might be something very basic.

For example, I had a researcher come to me recently because cells from my lab she had been given kept dying after they were retrieved from liquid nitrogen. The problem was that she was trying to culture the cells in a non-C02 environment with media that required CO2.

You shouldn't need to increase your incubator CO2 to 10% to buffer your medium (unless there is something wrong with the incubator). Did the DMEM medium contain Na bicarbonate and could the CO2 get into your cultures? This shouldn't be a problem with media buffered with HEPES as CO2 is not required. Are you having more luck with the HEPES buffered medium?