Media turning acidic quickly - (Feb/22/2015 )

I'm working with a new line of cells where the media is turning acidic very quickly.  I'm using DMEM (high glucose, pyruvate, l-glut) with FBS and insulin.  Can I supplement with Hepes to give better buffering capacity?

HEPES will buffer differently to bicarbonate, being used for low CO2 incubators, and doesn't (I think, could easily be wrong) work properly under CO2.  Check that you don't have a bacterial contamination, these can cause the medium to acidifiy quickly.

I don't think HEPES will solve your problem in CO2 incubator. To solve the problem, you must use more HEPES, but then this will disturb the cells. Maybe you need to use a different medium for that particular cell line.

How much sodium bicarbonate did you add? I add 2.5-3 g for 1 L.

Thanks for the replies.  The media is RPMI1640 that already has 2g/L sodium bicarb and I found a source saying to add 10mM Hepes so I'll see how that works.  There's no contamination as the media is clear, just yellowish after two days in culture.  Would it matter if they also require 15% FBS and 10U/ml insulin? 

Insulin and 15% FBS should be fine.