digitonin and nitr-5

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.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Cell Line; Chelating Agents; Cytosol; Digitonin; Egtazic Acid; Fibroblasts; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Manganese; Microscopy, Fluorescence; Photolysis; Rats; Signal Transduction; Spectrometry, Fluorescence; T-Lymphocytes; Tumor Cells, Cultured; Xanthenes; Zinc