Although not ideal, you can change media when doing CFSE assays. The stain is bound to your cells. The reason why it is not recommended is coz T cells form clusters when proliferating and any attempt to change media may break these clusters up, slowing proliferation. If you only change ¼- ½ the media volume, you could avoid disturbing the T cell clusters and get away with it.

Proliferation takes time and when and how long you should leave the drug for would depend on the mode of action of the drug. Ideally, you would want to leave it for the entire culture duration (5 days) to see if it has an impact on cell proliferation. It could be that the cells need the continued exposure or that the drug has an impact only once the cells have started actively proliferating (late time point, D3-D5). So if you add the drug early (D2, as you plan) and then remove it, you may not see an impact.

The only problem with this strategy is that prolonged exposure to the drug could be toxic to the cells. This is more of a concern when treating CFSE stained cells as CFSE itself is toxic. When you say your colleagues don’t change media for 5 days, is that when using the same drug? If so, you already know that the drug at the concentration used is not toxic to the cells. If not, you would have to first titrate the drug to identify a concentration that has an impact and is not killing your T cells.

Unfortunately, you may have no choice but to play with the different options. If you do know your drug’s mode of action and have an idea as to what it is supposed to do, you could use that to guide your experimental setup. If you do see an impact with prolonged exposure, you could then add it late or do wash out studies (add early, rinse and remove) to further tease out the drug’s mode of action.

Yes, changing the media when doing CFSE assays is not a problem. Care should be taken not to disrupt the ongoing proliferation.