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What is an acceptable CO2% range for growing cells in DMEM? - (Aug/17/2006 )

I am growing cells in DMEM medium in a 2nd-hand incubator. The CO2% levels jump between 4.7%-5.3%. is this an acceptable range? Thanks.


Just out of curiousity how are your cells doing in the incubator? Do they appear to grow alright (more viable than dead)? How quickly does the media color change (generally the media color will change if the CO2 levels are affecting the cells)?


Dear Nesta3,

The range I would imagine is easily acceptable if it only varied by 0.6%. If you are measuring via a Fyrite, then the level of accuracy is only 0.5%. In my experience not many scientist's I have come across even bother with Fyriting their incubators. They normally just go with what the levels say on the electronic display.
Since the development of IR CO2 Sensors, the drift we see in the modern incubators are no where near as large as 30 years ago, when Fyriting really had to be done on a weekly basis.

The art of experimentation is to reduce any error that can be easily avoided.....

Batch Testing of Serum as there are massive variations between batches.
Regular calibration of CO2
Regular calibration of Temperature
Regular calibration and use of Gilson pipettes
Using the same Tissue Culture plastics throughout a project
Regular Mycoplasma tseting of ALL cells, both primary and established lines.


Hi nesta3
It sounds OK. Normally the incubator should be pretty stable when its not being opened (the readout shouldn't vary by more than a point or two). Obviously the readout will drop when the door is opened and it will take a small amount of time to reach its set level after the door is closed.

It shouldn't be cycling up and down within a 0.6% range when its not in use and if this is happening you might want to get it looked at. I gather that it's been zeroed correctly and its inputting the correct amount of CO2. You can do a rough check with a fyrite if you have one, assuming its in good use.

We grow human fetal and placental cell lines and we actually routinely run our incubators at 5.5%. I know a couple of labs that run as high as 6%. My experience is most normal human cell lines (at least the ones we grow) grow better at slightly higher CO2 levels.


We grow cells in DMEM at 5% CO2 with a range of 0.5%.
If you notice that DMEM changes color "too much" (to evaluate this you could incubate only DMEM in a flask for 24h) and you can't solve your problem with the incubator you could add HEPES to DMEM.
But of course set the proper CO2% is better biggrin.gif !


Thanks for the reply. I am waiting to use the incubator until I am more sure about the CO2 range. What should I be looking for in the change of medium color? It usually takes the RAW monocyte cells a couple of days to change the DMEM a yellowish color in the incubator we already have running.


QUOTE (jamie419 @ Aug 17 2006, 08:47 AM)
Just out of curiousity how are your cells doing in the incubator? Do they appear to grow alright (more viable than dead)? How quickly does the media color change (generally the media color will change if the CO2 levels are affecting the cells)?


If the incubator is inputting too much CO2 (a lot more than 5%) the media will look yellowish. If it is inputting far too little CO2 the media will look purple/bright pink.

You can simply put a T25 flask with 5ml media into the incubator (loose lid) and check it after a few hours. It should be about the same color as a flask of media in another incubator you are confident is inputting 5% CO2.

As I said earlier, a half-decent incubator shouldn't cycle through a 0.6% range when it's not in use and I would be concerned that it's actual regulation is faulty (check to see if it's fan is working correctly). You can be confident your cells will grow fine b/w the range of 4.7 and 5.3% CO2 if that's what's actually in the incubator.

By the way, I never trust the CO2 readout unless I'm confident the incubator was zeroed correctly the last time it was shut down.


DMEM was originally formulated for use in 10% CO2 incubators. It is normally supplemented with 3.7 g/L Sodium Bicarbonate. Sodium Bicarbonate buffering for 5%CO2 incubators is normally around 2 g/L. I've done bicarbonate gradient experiments from 1-4 g/L sodium bicarbonate in a serum-free formulation in a 5%CO2 incubator where the best growth was 3.2 g/L. The cell line was a high density suspension cell line though.

You might be best off to pull a sample and measure the pH. The rule of thumb is the lower the incubator CO2% the lower the sodium bicarbonate concentration.

A reference would be Table 7.2 in Ian Freshney's "Culture of Animal Cells" 3rd Edition, pg 81.


Dear Nesta3,

If you are using RAW264.7 Mouse macrophages then you can use either DMEM or RPMI. Go back to the old method of using non filtered flask's and 25mM HEPES Buffered DMEM or RPMI. You do not need any CO2 using this media. At Wellcome research labs 15 years ago I was producing inducible Nitric Oxide Synthase for drug screening under exactly the conditions described above.
The only problem is that the HEPES buffer can only be used in "Closed systems" i.e. you cannot use 6/12/24/48/96 well plates, for plates you have to use Bicarbonate/CO2. Thus not ideal to use 2 differing buffers.
But needs must..... you should be growing the RAW's as a suspension culture in stirrer bottles. Most people do not as you need expensive stirrer platforms and spinner bottles. Some people just have non CO2 incubators and therefore have to use HEPES buffered media