Recently I performed concentration-dependent fluorescence studies with my active compound on THP-1 cells. I observed an increase in emission spectra for my active compound at the same time for untreated cells as well as lysis buffer.
Please find the figure below.
Looking forward to the explanation and way to carry out the experiment
It looks to me like all the spectra are about the same. There is no evidence that the fluorescence intensity increases with the compound concentration, nor does it depend on the presence of cells. The fluorescence may be coming from the lysis buffer, or it may be solvent Raman scattering. What is the excitation wavelength? What is in the lysis buffer? Have you tried just water?
I agree with Adam B Shapiro, you haven't observed an increase in fluorescence intensity, you have observed fluorescence in all your samples. Ditto to all his comments. What fluorescence are you trying to measure exactly? are you trying to look at changes in NAD/NADH or something else? To properly measure this you'd want you're lysis buffer only to be (ideally) around zero. It might be helpful to find a lysis buffer that doesn't fluoresce under these conditions.
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