I am trying to induce EMT in A549 cells by using TGF-beta as ligand. Currently, I am standardizing and it involves 24 hours of starvation and 48 hours of incubation with the ligand however by the end of the treatment the media (RPMI) turns yellow. Does it affect the transition? Should I add Hepes to avoid this pH drop?
This is normal, since your treatment is very long. And yes, you can try with Hepes, it is a good buffer. Additionally, use the maximum volume of medium allowed by your wells or dishes.
I agree with Josep, and would add that if possible, you can reduce the number of cells you plate, or increase the size of wells you use so that more media can be added.
For example, our lab has some studies in which we plate one million cells per well in a 12 well plate for a 48 hour subculture, refeeding after 24 hours. The media volume per well is 1.5 ml. We consider this to be standard for our experiments. Sometimes though, we want to treat cells with one or another drug for a longer time period, without refeeding. We will reduce the number of cells to 800,000/well and use up to 4 ml media per well. Our goal is to have 90% confluence by the day of the experiment or harvest. Based on your experience with your cell line and its growth pattern, you should be able to find a plating density and media volume that will prevent you from studying unintended starvation.
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