So recently my media has behaved very weirdly. Whenever I split the cells and transfer to a new flask containing orange media and put in the incubator, the media goes pink for several hours. And then it goes back to orange color again. I always check the cap to make sure it's loose but this has kept happening, only after I split the cells. If I feed the cells, there's no trouble at all. Does anybody have this issue before? I thought it was the HEPES so I remade it, pH it carefully but the issue remains.
Hi !
I would not care to much as long as it goes back to normal in a short time (1h-2h max), and as long as your cells do not suffer from this ...
If this color shift is only transiant , it can be the impact of the temperature on the wavelength your orange stuff absorbs. The second thing you must keep in mind is that in the incubator, cells are usually cultured with CO2 at a given percentage (5%) but your medium has almost no CO2 in it prior to its incubation in the incubator ... CO2 influences the PH, herby influences the pH tester color...
That's why if it goes back to normal color, its a good sign. But to ensure that your medium is not contaminated, you can put some antibiotic free medium in a culture flask inside the incubator ... if you are contaminated it should change color within 1 or 2 weeks...
Nevertheless, this pH and temperatures variations can have a bad impact on your experiments, so if you are not sure that this does not upset your cells , I would recommand you to pre heat the medium at 37°Cin a flask of your incubator so as to avoid any shock on the cells
Sorry for this long post, I hope it helps you with your problem !
G.A.
Nguyen,
If this is happening only when you split the cells, then I don't think it is just an issue of buffering with CO2, nor of the medium. Do you split with trypsin/EDTA? If so, do you neutralize the trypsin with FBS or medium containing FBS? This is very important. If you do not neutralize the trypsin adequately, then the trypsin goes on digesting and attacking the cell membranes, which is harmful to the cells. And the EDTA in the trypsin can also cause color change in the medium. It is very important to do a wash step after trypsinization to remove these substances from the cells. I know it is common to eliminate this step, and many cells can tolerate this, but over many passages this can become a problem and cause irritation to the cells.
Try washing the cells in complete medium (containing FBS) and centrifuging the cells, then resuspend and put into fresh flasks. (I use medium with 10% FBS at a ratio of 4-5 times the volume of trypsin used to be sure the trypsin is neutralized. You can also neutralize by first adding a small amount of pure FBS, then dilute with medium, especially if your trypsin is stronger than 0.05%) Hopefully, this will take care of the problem. You may still see a transient color shift due to the O2/CO2 balance, but hopefully, it will be much shorter duration, say 15 min.
Good luck,
Karen M. Peterson
Thank you all for your help. I also think it might be something to do with the trypsinization, but I do neutralize and wash out trypsin every time. I'll check again with other factors. Thanks again!
- Badges
- Science topic