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#1 Dukey

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Posted 02 April 2010 - 07:32 AM

Hey guys,

Got a real problem. Recently a whole cell line of mine was destroyed in a weekend when I purchased a new batch of media (same cat #). No sign of contamination, cells just round up and die slowly after adding the new media. The cells are grown in standard DMEM with 15% FBS and mercaptoethanol (ME). Because it was a new batch of media, I suspected glutamine degradation and so switched to glutamax and started with a fresh batch of frozen cells. All looked OK for a couple of passages until this week when I changed the media again and the cells did the same thing 24 h later. So it appears it is not the media. I know also that the serum is OK because I use it for all of my other cells and it is the same batch as I used previously when the cells were looking good.

So because the cells are dependent on ME and because it is the only supplement to the media that I myself add, I am pretty convinced now that my ME must be the cause of these issues. My ME was about a year old and stored at RT in a container. Luckily I had anticipated that and had a brand new one ready to go today.

Do you guys reckon this is sound reasoning? These cells are very picky and react very severely to changes in their environment. In the absence of ME, the cells will suffer dramatically. What could have happened to the ME? Degradation, cross contamination, reduction?

#2 Dukey

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Posted 04 April 2010 - 03:39 PM

Hey guys,

Got a real problem. Recently a whole cell line of mine was destroyed in a weekend when I purchased a new batch of media (same cat #). No sign of contamination, cells just round up and die slowly after adding the new media. The cells are grown in standard DMEM with 15% FBS and mercaptoethanol (ME). Because it was a new batch of media, I suspected glutamine degradation and so switched to glutamax and started with a fresh batch of frozen cells. All looked OK for a couple of passages until this week when I changed the media again and the cells did the same thing 24 h later. So it appears it is not the media. I know also that the serum is OK because I use it for all of my other cells and it is the same batch as I used previously when the cells were looking good.

So because the cells are dependent on ME and because it is the only supplement to the media that I myself add, I am pretty convinced now that my ME must be the cause of these issues. My ME was about a year old and stored at RT in a container. Luckily I had anticipated that and had a brand new one ready to go today.

Do you guys reckon this is sound reasoning? These cells are very picky and react very severely to changes in their environment. In the absence of ME, the cells will suffer dramatically. What could have happened to the ME? Degradation, cross contamination, reduction?


Looks like it could be a CO2 problem, culture media was close to pH 8.0 - oooooouch! God knows what the incubator is pumping in but I managed to titrate the CO2 so that the media pH is around 7.4. Painful to watch cells just die for needless reasons.

Edited by Dukey, 04 April 2010 - 03:40 PM.


#3 labrat612

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Posted 05 April 2010 - 10:28 AM

ooohh, man that sucks!

Your lab should invest in a Fyrite gas analyzer (or another type of gas analyzer). We use it to just make sure the setting in our incubator are what we think they are.

Glad you figured out your problem....


best,
Labrat612

#4 Dukey

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Posted 05 April 2010 - 12:52 PM

ooohh, man that sucks!

Your lab should invest in a Fyrite gas analyzer (or another type of gas analyzer). We use it to just make sure the setting in our incubator are what we think they are.

Glad you figured out your problem....


best,
Labrat612


Yeh I have used one before and all was good but I hadn't checked it in a while. The incubator is practically brand new too. I think because I use DMEM with the standard 3.7g/l bicarb any drop in the pH input (from 5%) can be quite dramatic for some cells. I have bumped the input up to 10% now to avoid any issues in the future. God knows why people use DMEM with 5%, the media is usually way to alkali! I just hope I can rescue some of the cells.




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