Recently I am repeating my mitochondrial calcium measurements with the protein sensor 4mt TNXL which is a FRET sensor working like the CFP/YFP-pair sensors (4mt D3cpv) but with a higher Kd. But the increase of signal ratio (FRET/CFP) is much lower than before with the same experiment settings, i.e. same microscope, exposure time, filter sets. I did some trouble shooting with the Ringer buffer used in experiment and concentration of free calcium in the solution, microscope settings, and used the other protein sensors for mito or cyto (R-Geco, intensiometric) . And I also replaced the cell line with Hek293 cells to see if it depends on the cell line.
In the end, it appears with both cell lines the cytosolic calcium influx following the addition of thapsigargin was too low (10-20% increase) to trigger mitochondrial uptake. Even if the extracellular calcium concentration was over 100mM, the increase of signal ratio from either cyto or mito sensor was only around 20%.
I thought that it might be some problem with thapsigargin and receptors. But after permeablization of cell memberane with ionomycin, the signal was still weak. And the other substances like hormone also did not work.
What else could be wrong?
Thanks for your attention!
100 Mm Ca2+ seems VERY high, you're probably disturbing the membrane potential or are causing osmotic stress depending on your other buffer components. I assume the pH of the buffer is identical, as is the temperature in which you do your experiments.
Can it be you have an issue with the expression of you sensor in the cells, I can imagine if you have a lower expression, the fluorescence signal will also be lower. You probably do keep your stuff in the dark and are aware photobleaching might be an issue? Did you try assesing the Ca2+ changes with a dye like FURA or FLUO? This might give you an idea if the difference is due to your sensor or due to the real Ca2+ influx.
Furthermore, it is not impossible that somehow your initial experiments were "wrong" and the lower Ca2+ you see now is "real". That's part of the reason we do repeats.
Good luck figuring this out!
Hi Zhang,
So, if I understand things correctly, your probe did work in previous experiments, but now there is nothing you can do to see a mitochondrial calcium response. It's hard to help remotely, and it seems that you have checked quite a few things. It is rather strange that you don't see mitochondrial calcium signals in response to agonist stimulation.
I would be tempted to go back to first principles and make sure your cells are showing cytosolic Ca2+ changes in response to thapsigargin and also hormonal stimulation. For this, I would use Fura2 or Fluo indicators. Once you have established the cells are showing appropriate cytosolic Ca2+ responses, you can check mitochondrial Ca2+ uptake in a simple, non-quantitative way using Rhod2. If this works, you could go return to using your FRET probe with the confidence of knowing your cells are responding ok. If there is no signal from your FRET sensor in cells that you know are responding it would suggest your probe is not working properly. You might then need to check your transfection or DNA.
As Koenraad pointed out, you really don't need to go up to 100 mM Ca2+. We never go beyond 2 - 5 mM in the most extreme experiments, and that gives a very robust Ca2+ influx response.
I hope this helps.
Best wishes,
Martin
Hi, Koenraad and Martin, thanks for replying to this question. I did some experiment with Hela cells with the same protein sensor, i.e. 4mtTNXL, 4mtD3cpv, R-Geco. And now it is quite clear that they all work properly, which means the problem should be the cellline-dependent. I also used Fura2 for once, the cells responded quite weakly just like the result from R-Geco experiment which is also a cytosolic calcium sensor. I have another question regarding this, does the Fret calcium sensor also answer to some other things like change of anion/ion concentration or osmotic pressure? The pH was controlled in the experiment, so I think it can not be changed that much.