3 replies to this topic

[Suhas]

My media composition includes 20mM NaOH. Why is NaOH added to media? What purpose does it serve?

[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.....

Suhas

[bob1]

The pink colour is the phenol red changing colour at different pH.  The MOPS and EPPS will also be buffering the solution, so you would probably  need to use those as well to counteract the NaOH.  I wouldn't rely on it only being there for pH, as CHO cells are quite fussy IIRC, but you could try it if you can afford wasting a few cells.

[HomeBrew]

How did you decide on 10 mM HEPES?  I'm guessing that 10 mM HEPES is inadequate to buffer 20 mM NaOH.  Did you add 1% Bufferall (which is sold at 100X) to your media before?

[Suhas]

HomeBrew, on 02 March 2011 - 06:55 PM, said:

How did you decide on 10 mM HEPES?  I'm guessing that 10 mM HEPES is inadequate to buffer 20 mM NaOH.  Did you add 1% Bufferall (which is sold at 100X) to your media before?

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?