Hi Marlon,

yollow color of RPMI media indicates an acidic pH. This you usually get, when your cells "used up" the media, this means, celular metabolites secreted into the media change the pH. But it can also be an indicator of a contamination, where metabolic products of bateria change the pH value.

So in the first case, it's quite normal, in the second case it's bad.

Cheers, Guido

But Doesnt matter that my RPMI was supplemented with bicarbonate and hepes?. Is supposed that this components keep the pH stable. Do you work with PBMC ?, if you do, do you make re-change the medium RPMI?. We know isnt contamination that make the medium change its color.

Thanks for your quick answer

Guido is correct. So if the cultures are yellow and the cells are not contaminated it must be due to cell metabolism. So it sound like the cells just need more care. Although your media is well buffered it may not be enough under your culture conditions. What is the PBMC concentration? In my hand I find that a PBMC concentration of 1-2x10e6 works well. How often do you change the RPMI? I do 50% medium changes every other day. How often do you split the cells? Do you add cytokines to the media such as IL-2 and if so what concentration do you use?

Hey Marlon! I have been working with PBMCs and I totally agree with Guido and Daniel. The yellow colour indicates fast metabolism of the cells. PBMCs have faster metabolic rate than most cell lines and the plating density also affects the amount of metabolites being produced. So, either you reduce the plating density or keep replenishing the media to have a healthy culture. A bacterial contamination check is strongly advised though. Happy Culturing !

I agree with everyone else, it is simply the pH changing as the cells use up the components in the media. Although you add HEPES to the media when you first use it, the pH will continue to change as the media sits around. You can either culture the cells at a lower density, or add a few mL of HEPES to your media each time you use it. Good luck!

I have the same experience as Marlon. The media turns yellow and from what I can observe there is no contamination. However I checked the pH of the culture and it is neutral. Can there be some other reason for the change in colour? Does the media change colour due to nutrient deprivation as such or is it only an effect of the pH change caused by the deprivation?

Turning yellow from red of RPMI is a clear indication of exhausted media i.e. acidic media; and this can happen when you do not change the media for long time and therefore PBMCs have utilized its components. In another case possible contamination. Make sure to change the 50% media regularly and keep appropriate cell density.

Guys, the question is: when I prepare the media for use outside CO2 incubator I add HEPES buffer to the media with concentration 15-25 mM, the question is, should the media (containing Phenol RED) instantly turn yellowish-red (pH 7.2-7.3)?

Anette, Marlon, I tried to titre the HEPES buffer like it was said in the protocol till 7.2-7.4 pH, but upon achieving 7.2 adding 1M NaOH (using previous dosages) was not making a lot of difference, so I did not continue to add more...

Guys, the question is: when I prepare the media for use outside CO2 incubator I add HEPES buffer to the media with concentration 15-25 mM, the question is, should the media (containing Phenol RED) instantly turn yellowish-red (pH 7.2-7.3)?

Anette, Marlon, I tried to titre the HEPES buffer like it was said in the protocol till 7.2-7.4 pH, but upon achieving 7.2 adding 1M NaOH (using previous dosages) was not making a lot of difference, so I did not continue to add more...

Aneesh Nair I have been using RPMI in my lab for an experiment with PBMCs in a short-term cultures. Nothing out of ordinary culturing experiment. However, our cell suspension and the medium are turning to a very dark red and cells are not clonally expanding. Hove you seen this problem before? How do you troubleshoot it?

Thanks a lot.