Photochemically generated cytosolic calcium pulses and their detection by fluo-3

TLDR

Fluo‑3 is a fluorescent Ca²⁺ indicator excitable at visible wavelengths that has been tested in living cells. Fluo‑3 can be microinjected into fibroblasts with photolabile compounds for photorelease experiments and calibrated using ionophores and heavy metal ions to quantify intracellular Ca²⁺. Loading fibroblasts and lymphocytes with the pentaacetoxymethyl ester of fluo‑3 yields intracellular dye that reports agonist‑induced Ca²⁺ changes, enabling continuous monitoring without interfering with UV‑sensitive caged compounds. Chem.

Abstract

Fluo-3, one member of a family of new fluorescent Ca2+ indicators excitable at wavelengths in the visible (Minta, A., Kao, J. P. Y., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171-8178), has been tested in living cells. We demonstrate that fluo-3 can be loaded into fibroblasts and lymphocytes by incubation with the pentaacetoxymethyl ester of the dye and that the ester is hydrolyzed intracellularly to yield genuine fluo-3 capable of indicating changes in [Ca2+]i induced by agonist stimulation. Fluo-3 can also be microinjected into fibroblasts along with photolabile compounds such as nitr-5 and caged inositol trisphosphate for photorelease experiments. Fluo-3 permits continuous monitoring of [Ca2+]i without interference with use of UV-sensitive caged compounds. A procedure for combined use of ionophore and heavy metal ions in end-of-experiment calibration of fluo-3 intensities to give [Ca2+]i is also described.