Media - a beautiful pink!
Posted 02 March 2011 - 09:43 AM
[u]So the issue is...
My Ham's F-12 media [for CHO cells] apart from 10% FBS and 1% PS also had 20mM NaOH and 1% bufferall [sigma]. Bufferall has HEPES, MOPS and EPPS and i guess maintains the pH.
Sigma stopped manufacturing of bufferall. So i started using 10mM HEPES instead.
But when i add 20mM NaOH, my media turns a beautiful pink and the pH is around 11.
If NaOH is only added to pH the solution, i can skip it right? But i'm not sure if its serving any other purpose here that could affect my cell growth.
I have two types of cells - CHO cells transfected with myoglobin gene and CHO cells that do not produce myoglobin but have an antibiotic resistance gene [geneticin]
any ideas? i need to prepare media like right now! plz plz plz.....
Posted 02 March 2011 - 02:49 PM
Posted 02 March 2011 - 06:55 PM
Posted 03 March 2011 - 07:49 AM
For now... i added all the media constituents except NaOH. Since i don't have any bufferall left, I added 10mM HEPES as a buffering agent. The cells seemed to be happy as of now. But not sure... sometimes things go wrong only after the 2nd or 3rd split
I used to use 1% of the 100X bufferall before. Since its production stopped, and no similar product was available, i just used a buffering agent that the rest of the lab used for other cells- 10mM HEPES. I checked with sigma and they told me bufferall had HEPES, MOPS, EPPS. I can make my own buffering agent by adding all the three buffers but i'm not sure if it'll still counteract the effect of NaOH. 20mM NaOH has a pH of around 12 or something. And these buffering agents have a buffering capacity around 7 - 8.5. Will that be enough?