Hi,

If you do have a GFP tag, one of the rhodamine dyes may be the best choice, probably Rhod-2 or x-Rhod. The Fluorescence Handbook, from formerly Molecular Probes, now ThermoFisher, is probably still the most useful text for making these kind of decisions (https://www.thermofisher.com/nl/en/home/references/molecular-probes-the-handbook/indicators-for-ca2-mg2-zn2-and-other-metal-ions/fluorescent-ca2-indicators-excited-with-visible-light.html).

Best,

Michiel

Hi,

The key difference is that Fura is a dual excitation dye, that is, you have to excite it at two different wavelengths to be able to do a ratiometric measurement, for which it is designed. While that is great for quantitative measurements, it usually doesn't work with a confocal. They are not set up for dual-excitation measurements, and the wavelengths, 340 (or 360) and 380nm, are not available on a confocal. That leaves you with single excitation dyes like Fluo. Which dye to use depends on the expected calcium concentration. Fluo 3 is very sensitive, for concentrations between 0.1 and 1 microM. 

You could do spectroscopy, not sure what informatie you would get out of it that is useful for the confocal experiments.

All the best,

Michiel

Hi, 

I agree with Michiel, Fura is not a good option when you want to use it in confocal,but in your case, maybe fluo 3 is not the best choice, either. you have tagged a GFP with the receptor, the GFP and Fluo 3 almost have the same excitation wavelength (around 480 nm), which means when you record the Calcium signal, you also record some signal form GFP, that is not a perfect condition for your experiment. maybe you can try to tagged your receptor with other tags like RFP, which is excited at 560 nm.

Best 

Wenying

Hi,

I use Fluo-4 to mark the intracellular calcium adherent cardiomyocytes in fluorescence microscopy. I use a kit that requires 45 minutes of incubation and the fluorophore does not need to be removed and the rinsing is not necessary, which allows not to stress cells.

Fluo-4 is really better than Fura-2, Fluo-4 allows to have a greater intensity. It also works very well in spectrophotometry on cells in suspension.

Good luck,

Hélène

https://www.thermofisher.com/fr/fr/home/references/molecular-probes-the-handbook/indicators-for-ca2-mg2-zn2-and-other-metal-ions/fluorescent-ca2-indicators-excited-with-visible-light.html

Hi,

If you do have a GFP tag, one of the rhodamine dyes may be the best choice, probably Rhod-2 or x-Rhod. The Fluorescence Handbook, from formerly Molecular Probes, now ThermoFisher, is probably still the most useful text for making these kind of decisions (https://www.thermofisher.com/nl/en/home/references/molecular-probes-the-handbook/indicators-for-ca2-mg2-zn2-and-other-metal-ions/fluorescent-ca2-indicators-excited-with-visible-light.html).

Best,

Michiel

Thank you for all. I think fluo-3 is better so I will use flag-tagged protein. Antibody which i have for confocal microscopy against flag exhibit red color so red color calcium dye is also not for good to use. 

I know fluo-4 is better than fluo-3 but fluo-3 is cheaper so I prefer fluo-3.

And I will analyze calcium concentration using confocal microscopy and microplate reader. If I use microplate reader, should i use dual excitation ratiometry dye?

Then which one is better? fluo-3 and fura-2? or fluo-8? or any other dyes?

Please help me. There is no one who did calcium works in my university.