cells in culture

Jayakumar, R via methods%40net.bio.net (by R.Jayakumar At roswellpark.org)
Fri Jan 26 18:19:08 EST 2007


Before blaming anything else, have you checked for mycoplasma
contamination?  Take care to grow the cells to near 90% conluency for at
least 4 days before taking the supernatant for check.  Use a DNA PCR
based kit.  
   Also check the CO2 levels in the incubator (not the digital display
on the incubator which does not show the actual content of CO2 but is
only a calibrated value), but measure it manually using Fyrite which is
the most accurate way of estimating CO2.  I had a similar situation once
when my cells started dying mysteriously on the 2nd to the 3rd day of
passage.  The CO2 showed 5% CO2 and everything else looked normal.  But
the pH color gave me the indication that something is wrong with the
CO2. Turned out he incubator was new and had never been calibrated with
a Fyrite instrument. After the calibration the cells started growing
normally.  
   Are you and your colleague using the same incubator or hood??
   If DMEM is from GIBCO, I think you have one of the best and most
reliable medium about.
Best of luck
Jai
 


-----Original Message-----
From: methods-bounces At oat.bio.indiana.edu
[mailto:methods-bounces At oat.bio.indiana.edu] On Behalf Of Tom Anderson
Sent: Friday, January 26, 2007 11:22 AM
To: methods At magpie.bio.indiana.edu
Subject: Re: cells in culture

On Thu, 25 Jan 2007, Pow Joshi wrote:

> Thanks, Wolfgang....that histoire was comforting, knowing that I 
> was'nt the only one....

I just spoke to one of my colleagues, whose HEKs are also mysteriously
dying on her. Is it possible there's an iffy batch of medium about?
She's using some sort of Gibco DMEM.

My other thought was that it might be something related to the christmas
holiday; if, for example, your insitute's water purification system was
switched off over the break, it might have accumulated some sort of
contamination, which could now be present in any media, PBS, etc that
you make up locally. This would be unusual, and it would be odd for it
to affect cells as robust as HEKs, but it has happened to people here.
But then you'd expect other people to be having the problem as well.

tom

--
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London
WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e)
thomas.anderson At ucl.ac.uk

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