BCECF AM ester is a widely-used fluorescent indicator for estimating intracellular pH.[1],[2] This membrane-permeant compound is modified by intracellular esterases to produce BCECF, a polar fluorescein derivative that is retained by cells. Intracellular BCECF is stable and has an efflux half-life of greater than two hours.[3] Intracellular pH measurements are made by ratio imaging, which involves determining the pH-dependent ratio of emission intensity (detected at 535 nm) when the dye is excited at 490 nm vs. the emission intensity when excited at 440 nm.[2],[4] This approach is amenable to either spectrofluorometry or flow cytometry.[2],[5] BCECF AM can also be used to investigate intracellular changes in other ions, including potassium.[6]
Reference:
[1]. Rink, T.J., Tsien, R.Y., and Pozzan, T. Cytoplasmic pH and free Mg2+ in lymphocytes. Journal of Cell Biology 95(1), 189-196 (1982).
[2]. Ozkan, P., and Mutharasan, R. A rapid method for measuring intracellular pH using BCECF-AM. Biochimica et Biophysica Acta 1572(1), 143-148 (2002).
[3]. Dive, C., Cox, H., Watson, J.V., et al. Polar fluorescein derivatives as improved substrate probes for flow cytoenzymological assay of cellular esterases. Mol.Cell Probe 2(2), 131-145 (1988).
[4]. O'Connor, N., and Silver, R.B. Ratio imaging: Practical considerations for measuring intracellular Ca2+ and pH in living cells. Methods Cell Biol. 81, 415-433 (2007).
[5]. Chow, S., Hedley, D., and Tannock, I. Flow cytometric calibration of intracellular pH measurements in viable cells using mixtures of weak acids and bases. Cytometry 24, 360-367 (1996).
[6]. Balkay, L., Márián, T., Emri, M., et al. Flow cytometric determination of intracellular free potassium concentration. Cytometry 28, 42-49 (1997).