Fluo-4 is a great non-ratiometric dye

While Fluo-4 is a good dye, the excitation spectrum overlaps too much with that of YFP to be useful. Do you have access to a high speed monochromator? If so, then Fura-2 would work well as it excites in the UV range, is ratiometric (excitation at 340 nm and 380 nm, and emitting at 510 nm), and therefore its excitation does not overlap with that of YFP. If you don't then Fura may not be your best bet, though its binding kinetics are ideal for this type of study. Do you also need to worry about visualizing YFP simultaneously with calcium measurements or would measuring YFP at the end of the experiment be sufficient?

Wait, what is the YFP fused to? If it is attached to Calmodulin or MYLK I think you can use the YFP directly as your indicator.

Thank you for your help. I can't use FURA-2 since I do not have dual excitation setup. YFP is not fused to Calmodulin or MYLK1. I thought about X-Rhod1 or calcium Crimson. I never used these dyes myself and was wondering about their biophysical properties: fast bleaching, easy loading, good sensitivity etc...

To answer to Brian, yes I need to image YFP to distinguish between the cells expressing/not expressing YFP

You can use Indo-1. That is UV excited and the emission is around 420 nm. I previously used that dye with CFP and YFP. You can use the dye for ratio metric Calcium imaging. In my experience, red calcium dye is relatively difficult to use. It depends on the cell types that you can success the imaging with red dye.

You can use Indo-1 for single wavelength imaging too. And also, you can use Indo-5F too. The Kd of Indo-1 is around 100-200 nM, and the Kd of Indo-5F is around 500-600 nM. You can choose your suitable dye. The Kd of Indo-1 is almost same as Fura-2. Please make sure to purchase Indo-1-AM or Indo-5F-AM.

Maybe "Asante Calcium Red AM", but only in non-ratiometric manner with an emission at 650nm. I myself load cells with Fluo-4-AM, but since the emission wavelength is similar to YFP, it is, of course, not possible. If you find any other possibility, you have not to forget about the kd for calcium.

Hello Dmitry, I'm interested in your suggested dye "Asante Calcium Red AM". Have you used that dye? If yes, could you let me know your conditions (Cell type, loading condition, and the result etc.)? I want to try it!

Muriel, I wrote red dye is difficult in previous message but reminded that fura-red worked well in HeLa cells.

Dear Toru Matsu-ura, to be honest I have no personal experience with this dye. Maybe this publication will help you http://www.ncbi.nlm.nih.gov/pubmed/24017967

And here it has been used in cardiomyocytes : http://www.ncbi.nlm.nih.gov/pubmed/21575569 and here are manufacturer's data : http://www.teflabs.com/Portals/44052/docs/AsanteCalciumRed-Info%20Packet.pdf#page=2&zoom=auto,69,679 and also here http://www.teflabs.com/Portals/44052/docs/AsanteCalciumRed-Info%20Packet.pdf . It is available from TEFLABS as Potassium salt and AM-Ester. I myself investigate calcium signals in sperm cells. Regards, Dmitry

Maybe you can find especially in the paper from Biophys. J. conditions to use this dye ?!

Dear Muriel,

I agree with all the suggestions above, however because fura-2 and indo-1 are excited in the UV, you will need a scope with UV-compatible optics and objectives (because they are more expensive; generic scopes may not have these). Note, the Indo family are also dual *emission* (fura-2 is dual *excitation*). Only a very high temporal resolution system will be able to alternate wavelengths fast enough to capture a spark (over in ~200ms). You'd be better off with a single wavelength set-up unless you have this facility (or a beam splitter for Indo-1).

The Asante Red. No practical experience myself, but the signal:noise doesn't look spectacular and if you are looking for unitary events like sparks then s:n is everything.

Toru mentioned Fura Red and it is ok for large responses but I would question its usefulness for small sparks because FR fluorescence *decreases* with Ca2+-binding.

Another word of warning with red dyes: some of the rhod family (like rhod-2) readily compartmentalizes in mitochondria (since they are positively charged in their AM form). Great for measuring mitochondrial Ca2+, but sucks for measuring the cytosolic signals! This IS cell-type specific, so you always need to check where your dyes are in any 'new' cell model you are trying. You might be lucky.

Not a great deal of help, but some things to think about.

Is there a special reason why you have to tag with YFP ? Why not mCherry ?

If there is no special reason, it is best to produce "another plasmid" and to use FLUO-4-AM. To my experience it is one of the best dyes. I measure changes in calcium in human sperm where 1 calcium ion makes a difference of 2.5 nM in concentration and even under these conditions I have wonderful signal to noise ratios !

I agree with Dmitry.

Also F4 is indeed good, but there is also Fluo-2 (Teflabs) which is sold by other companies under the name of "Fluo-8" (but much more expensive than Teflabs who invented it!). It is twice as bright as fluo-4 (and 4x brighter than fluo-3).

Again, some cells compartmentalize some of these dyes so fluo-4 may be less or more cytosolic than fluo-8, depending on cell type.

Thank you all for your very helpful answers. I am not able to use dual excition , and appreciate that Fluo-4am is altogether the best calcium indicator for calcium sparks analysis. We are thinking of sorting cells by FACS, and then if all are YFP positive, we do not need anymore to worry about YFP fluorescence. The problem I have is that we have a mix population YFP/no YFP, so that once loaded with Fluo-4am we cannot know anymore which one are YFP positive.

Are you able to infect with virus or make a stable line? Similarly, all cells will then be YFP-positive?

Yes, Anthony, this true ! Lentiviral transduction with HIV-1 based lentivirus gives at least for hippocampal neurons a transduction rate of 80-95%. This works very well. But you need an S2 lab !

I combined the Calcium Orange (life technologies) with vectors expressing EYFP. I used the laser 595 line to excite calcium orange. With calcium orange you don`t need to do ratiometric calculation. you can measure calcium transients directly by calculating the ΔF/F0

If you happen to have access to a two-photon microscope, you should be able to separate Fluo-4 from YFP by excitation wavelength.

To come back once again to Asante Red - our lab tried some month ago and it was not working for us. Only noise. By the way there are also Cal520-AM and FluoForte-AM as alternative calcium dyes available.

Perhaps loading with the (AM form) of the rhod familly of Ca2+ dyes:  (rhod-2, x-rhod-1, X-rhod-5F). We used this to image Ca2+ changes (mitochondria) in YFP-expressing terminals (below). 

http://www.ncbi.nlm.nih.gov/pubmed/16613870