I have a cell that expresses a luciferase protein upon adding substrate. Our luciferase protein has an excitation wavelength of 488 nm and therefore it is supposed to be able to excite a yellow fluorescent protein. Plate reader (in luminescence mode) can be used to check if such a system works. Because of some limitations, I highly prefer other techniques, if it is possible. My question: can I use FACS (flow cytometry) to test the system? Using FACS machine, in our case FACSMelody, I can see if the cell expresses YFP protein by a combination of laser 488 nm (for excitation ) and filter 530 nm (for emission). Now, I want to use our luciferase protein instead of the laser 488 nm to excite YFP. if I use a FACS machine that has laser 488 nm, but (i) laser 488 nm is combined with filter 613 nm (not in the range of YFP emission wavelength), and (ii) laser 561 nm is combined with filter 530 nm,
can I say I am seeing the output of my system in 530 nm histogram graph, although YFP is excited by 488 nm laser (but its emission wavelength can not be detected because there is no detectable filter combined with laser 488 nm in the machine)?
Gita Naseri , it is difficult to answer your question without detailed information about fluorescence, absorption spectra of components under study. I'm sure that you know them. I propose you to draw these spectra on the same plot and put it for public access. In such conditions your question will be much clearer for audience, and sometimes you will understand the answer even without help of others. Very often good formulated question contains inside it correct answer...
I think it is hard to realize. Because bioluminescence signal is weaker than fluorescence， as we can easily see GFP with a microscope, however we can't see the bioluminescence. And according to PMID: 8187580
- DOI: 10.1002/cyto.990150305, Firefly luciferase is not strong enough to be sorted by FACS.