Flow cytometric determination of intracellular free potassium concentration

Abstract

A flow cytometric assay has been developed for determination of intracellular free potassium concentration ([K+]i). Investigated cells, loaded with the fluorescent pH indicator 2',7bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF), are incubated in the presence of nigericin, and the intracellular pH is measured. The ionophore maintains the same ratio value [H+]/[K+] on both sides of the cytoplasmic membrane, so [K+]i can be evaluated from the measured intracellular pH (pHi) and the known parameters of the buffer. Application of the method revealed that the intracellular potassium concentration is significantly higher in lymphocytes than in proliferating cells of HUT-78, U266, and JY cell lines. A surprisingly low (60 mM) [K+]i concentration was observed with sperm cells of common carp. This method allows measurements on individual cells, provides data of excellent statistics, and still does not require large amounts of material. These features offer remarkable advantages over other techniques used for intracellular K+ measurements, such as steady-state fluorescence, atom absorption photometry, or energy-dispersive x-ray analysis.