Hello Alena Gorb If I understood correctly, the F0 is completely untreated cells so that is your negative control. And yes all should normalize to it. But you also have to account for treatment procedure when measuring the treatment effect. So the intensities for treated cells should also be normalized by "sham" or vehicle only intensities to determine the actual effects of adding cytokine alone or cytokine with inhibitor. Unless your F0 cells have had the vehicle only treatment also. So F0 is background or basal level subtraction for all , and sham/vehicle only will normalize any effects the actual protocol for transfection/transfer may have had. hope this helps.

Hi Rabeah Al-Temaimi

Thank you very much for your response! So just to clarify, I was only able to calculate F0 for cells that were acutely treated with KCl (which is our positive control) 1 min after the imaging was started. So I used the mean intensity before 1 min as F0 for these KCl-treated cells only. For the rest of the conditions (cytokine, cytokine+inhibitor, and vehicle), the cells were pre-treated with these agents 24 hours before the imaging, so there wasn't any acute addition of any agents during the imaging. I also do not have any completely untreated cells to use as F0, as you've suggested, so I am not sure what would I use as F0 (if anything) for my 24 hrs pre-treated cells? But your suggestion on using the vehicle for normalisation of cytokine treatment makes a lot of sense and I will discuss it with my supervisor, so thank you so much for it, it is very helpful!