I've been using Fluo-4 am (invitrogen) to image cytosolic calcium level and I noticed calcium level in nuclear is higher than cytosolic calcium.
Can I compare the level of calcium between cytoplasm and nucleoplasm based on Fluo-4 am imaging?
Hi Chi Woo,
Because Fluo-4 intensity depends on both [Ca2+] and the concentration of Fluo-4 (and light path), you can not compare cytoplasm vs. nucleoplasm [Ca2+] based on Fluo-4 (or any other non-ratioable dye) fluorescence. For example, higher fluorescence in the nucleus compared to cytoplasm might be due to preferential accumulation/binding of Fluo-4 in the nucleus (rather than higher [Ca2+] in the nucleus.
All the best,
Gavriel
I can not compare cytoplasm vs nucleoplasm but at least I can say that there is calcium influx into nuclei using Fluo-4 imaging, right?
I have to disagree with Gavriel. It is possible, but non-trivial. Obviously, you would need to be aware that the Kd and dynamic range of the dye depends on the cellular microenvironment and must be calibrated independently for the different compartments. Also, you would need to take some care to insure that the dye is reliably saturated, which may be difficult to accomplish.
Please refer to the following paper for more detail on the suitability of various indicators for cytoplasmic and nuclear [Ca2+] estimation:
D. Thomas, S.C. Tovey, T.J. Collins, M.D. Bootman, M.J. Berridge, P. Lipp, A comparison of fluorescent Ca2+indicator properties and their use in measuring elementary and global Ca2+signals, Cell Calcium, Volume 28, Issue 4, October 2000, Pages 213-223, ISSN 0143-4160, http://dx.doi.org/10.1054/ceca.2000.0152.
Shane, thank you for citing our paper. I completely agree with what you wrote.
Chi, it is possible to use single-wavelength indicators to measure calcium in many different cellular compartments simultaneously. My group has done that for cytosol, nuclei and mitochondria over the years, but as Shane mentioned you need to take care.
There is a long-running literature concerning differences between nuclear and cytosolic Ca2+ concentrations in lots of cell types. But, in many instances these observations have been simply due to lack of appropriate indicator calibration.
A common way of presenting data when using single-wavelength indicators is F/F0. Where F is the indicator fluorescence during the experimental run, and F0 is the starting fluorescence. Providing that background correction is performed, this can help to control for variations of indicator loading between cells, but it does not correct for the different Kd and dynamic ranges of indicators in alternative cellular compartments that Shane mentioned.
If you see a change in F/F0 as you run your experiment, and the change occurs in both the cytosol and nuclear compartments then it would be reasonable to suggest that there is a Ca2+ response in both locations. However, you cannot simply compare the amplitude of the F/F0 data.
Happy to offer more help, if you need it. As, I am sure, can Shane too.
Best of success with your experiments. Martin
Martin,
Thanks for elaborating.
Chi,
As Martin suggest, if all you require is a qualitative indicator then F/F0 would be sufficient to show nuclear Ca2+ influx. This is a relatively easy measurement to make. If you need a quantitative measure, then Martin's paper is a good place to start. From there you should be able to find several other good, relevant papers. I have some experience in that area as well, and would be happy to answer any questions you have after consulting the relevant papers.
As Gavriel mentioned, in some cases a ratio metric dye offers certain advantages. Additionally, many Ca2+ indicators can be used in combination with fluorescence lifetime imaging to circumvent the uncertainty introduced by dye concentration. I have less practical experience here but I may still be able to direct you towards relevant literature. However, these methods typically require different hardware than one normally uses when using fluo-4 as a dynamic range Ca2+ indicator. However, the hardware requirements for ratiometric imaging of the dual wavelength emission dye Asante Calcium Red would probably be the most similar.
cheers,
Shane
I would rather like to ask a question in continuation to this conversation that do we know that Fluo-4 am is indicating free calcium or calcium bound to different proteins as well ??
Thanks in advance
Fluo-4 and other fluorescent calcium indicators measure the free calcium concentration. If you want to estimate the total calcium concentration then you'll need to measure the calcium buffering capacity. A 1992 paper from Neher provides a good starting point: Article Calcium gradients and buffers in bovine chromaffin cells
The method's have evolved a bit since they were introduced by Neher but his method and explanation are probably the easiest to follow.
If you want to know the calcium bound to a specific protein of interest, then you would also need to know the Kd and concentration of your protein. Unfortunately, that information is not generally available.
HI Neelima,
Just to add to Shane's information: another way to go to measure total calcium within a cell/compartment is to use radioactive 45Ca2+ (something I did a lot of in my PhD, many years ago!). If you know the specific activity of the 45Ca2+ and the cell/compartment volume, you can calculate the total calcium. This usually requires a large populations of cells or purified organelles though.
Whilst calcium indicators don't measure calcium bound to proteins or lipids etc., they may bind to proteins themselves, which may alter their properties such as affinity and distribution (DOI: 10.1016/j.ceca.2018.04.005). We make some assumptions when we use calcium indicators, and even more when we calibrate their fluorescence into calcium concentration.
Best wishes,
Martin
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