Hi, I am about to do calcein imaging to measure cell volume under iso and hypo conditions. I checked online and noticed that calcein can interact with calcium. In order to prevent any undesired effects from calcium binding, should I prepare loading solution and bath solution without calcium? Thanks!
Hi Chen Kang,
I guess you'll do this in vitro. Modifying calcium can affect cell adhesion (cell to cell, or cell to substrate) so I would recommend not doing that. Why are you using calcein? Have you considered other possibilities, such as cytoplasmic or membrane GFP?
Thanks! Good luck!
Thanks for your info. Because calcein is our available tool to measure cell volume changes at this time point. I might not state clearly about my concerns. I still use the regular DMEM plus specific concentration of calcein to incubate with the cells and then when I use different bath solution to perfuse the cells mounted on the microscope stage, the bath solutions do not have calcium since I checked online calcein can also interact with calcium which would affect the signals emitting from it.
Removing calcium from the bathing solution is not a good idea. Cells lose their physiology and attachment properties in either low calcium or calcium free condition. I believe, Chen was confused with the principle of using calcein in measuring change in cell volume. Calcein-AM when after cellular permeabilization is cleaved by nonspecific esterases form free calcein, which when bound to calcium gives a fluorescent color. By removing extracellular calcium in bathing media will have nothing to do with the availability of intracellular free calcium and its availability to calcein to produce detectable fluorescence.
After loading with the dye the cells need to be washed thoroughly with the isotonic perfusion buffer to get rid of external dye molecules and to ascertain complete deesterification of the dye inside cells adequate time should be allowed before starting an fluorescence ratiometric experiment. To avoid unwanted photobleaching of the dye, frequency of exposure per unit time may be reduced.
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