Hi,
is it possible to quantify the intracellular calcium concentration (in adult mouse cardiomyocytes) using the Ca2+ indicator Fluo-4?
Thank you very much.
According to a report the loading of Ca 2+ indicator Fluo-4 into cells and binding of anti-rabies sdAb-Fc antibody to FcεRI receptors is performed simultaneously to reduce the damage to RBL cells and ... Its main drawback is that it is not a ratiometric dye; hence, quantifying the absolute levels of basal calcium levels using this dye is not possible.
Fluo-4 is not ratiometric, and thus you are only able to measure changes in calcium concentration. To be able to quantify the calcium concentration from those relative changes, you need a fix point to base such an estimate on. For example you could saturate the indicator by flooding your cells with calcium. You then know Fmax, if you know the maximum change in fluorescence (Fmax/Fmin) and the dissociation constant of Fluo-4, you can use that to estimate the absolute calcium concentration. The formalism is described in detail in the linked paper from Maravall et al.
Article Estimating Intracellular Calcium Concentrations and Bufferin...
An alternative would be to replace Fluo-4 with the spectrally very similar indicator Oregon-Green 488 BAPTA-1 (OGB-1) and use fluorescence lifetime imaging to quantify the calcium concentration.
Both methods have caveats, but so does the use of ratiometric indicators such as Fura-2.
Good luck!
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