Help: HEPES in CO2 incubator? - (Jun/20/2007 )

Hi, I'm using a DMEM medium with 25mM of HEPES (44mM of Sodium bicarbonate) to grow some fibroblasts in a 5% CO2 incubator and my cells really are not doing so well. I was wondering... if it's because there is 25 mM HEPES in my medium?I checked out some other info on HEPES which tells me that to adequately buffer the system with the presence of CO2 with HEPES, I should have 2X the amount of the Bicarbonate that I'm using. Can anyone help me? Should I then add in another 44X2-25= 63mM of HEPES? I thought above 50mM HEPES might be toxic to cells?

much thanks in advance

I dont think buffer components are that critical for fibroblasts. Sodium bicarbonate or 25 mM HEPES should do fine. Besure to seed enough cells, or primary fb would not grow well in some cases.