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

• Published in: Analytical biochemistry Vol. 567; pp. 8 - 13
• Main Authors: Rana, Priyanka S., Gibbons, Brandon A., Vereninov, Alexey A., Yurinskaya, Valentina E., Clements, Robert J., Model, Timothy A., Model, Michael A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.02.2019
• Subjects: Asante potassium green; Calibration; Cell volume; DiBAC4; Flow cytometry; Gramicidin; Intracellular potassium; ION potassium green; Ionophores; Valinomycin; Valinomycin; Flow cytometry; A; DiBAC4; ION potassium green; G; Calibration; M; Ionophores; N; Gramicidin; Asante potassium green; V; APG; Cell volume; Intracellular potassium; DiBAC
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2018.11.024

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. •General principles for calibrating intracellular ion probes are suggested.•The best treatment for intracellular calibration of K+ probes is suggested.•To ensure correct calibration of APG-4, cell swelling must be prevented.•APG-2 is nonspecific for K+ and reports the total concentration of monovalent ions.