Hello guys, I am doing some experiments which involve increase of cytosolic calcium. At present i do not know what is the source of this increased calcium in cytosol. So i just wanted to confirm what are the best methods/dyes to study the organelle (mitochondria or ER) specific calcium release.
Also i am using Fluo-3AM for calcium measurement and i have observed that increase in cytosolic calcium was detected after 6 hours of stimulation which continues to rise up to 24 hours (by flow cytometry). I used Thapsigargin as Positive control.However, I am being told that fluo-3AM signal is not only dependent on calcium concentration but also on cell volume and granularity.I was recommended to do the ratio-metric analysis. So please suggest is it possible to do the ratio-metric analysis at longer time points after stimulation. I hope this much experimental is enough for some description will help you to give some good suggestions. Thank you in advance.
Dear Mohammad, Normally probe tend to leave the cytosol. It can be taken up by organelles or be extruded via transporters. Therefore probe signal decreases as a function of time in most cell types i have worked with. I would do the stimulation, then wait, and then load with fura-2 am or another ratio probe, and then compare the signal with an unstimulated control. Best wishes for your exp.
Thank you sir for your suggestion. But i want to know which method is better to analyze the calcium efflux into cytosol. Is it better to use fluorimetry based methods or microscpy. Thank you once again
If you want to be sure that you actually get a flourescent signal from the cytosol, and not from organelles, microscopy analysis would be the safest bet. And classical video microscopy with a xenon lamp, fura-2 and dual emission filters would probably be ideal.
I also recommend Fura-2 AM for a ratiometric dye. The dye can be retained in some cell types for many hours, but as Birger mentioned it can be pumped out of certain cells. Probenecid can be used to prevent dye pump-out but will have to be controlled for. I prefer not to use probenecid.
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