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.