Calibration and characterization of intracellular Asante Potassium Green probes, APG-2 and APG-4

Abstract

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular and extracellular concentrations of the measured ion must be made equal. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two potassium probes, APG-4 and APG-2. APG-4 shows good sensitivity to potassium but its fluorescence is sensitive to cell volume. Because ionophores cause cell swelling, calibration buffers had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured at 132 mM and 118 mM, respectively. The other tested probe, APG-2, is nonselective for cations; this is, however, a potentially useful property because the sum [K⁺] + [Na⁺] determines the amount of intracellular water.

Keywords: Asante potassium green; Calibration; Cell volume; DiBAC(4)(3); Flow cytometry; Gramicidin; ION potassium green; Intracellular potassium; Ionophores; Valinomycin.