Photochemically generated cytosolic calcium pulses and their detection by fluo-3.
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.
Kao
J P
JP
Department of Physiology-Anatomy, University of California, Berkeley 94720.
Harootunian
A T
AT
Tsien
R Y
RY
D000814:Aniline Compounds; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents; D003600:Cytosol / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010782:Photolysis; D051381:Rats; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014966:Xanthenes; D015032:Zinc / Q000494:pharmacology
D000814:Aniline Compounds; D001572:Benzofurans; D002614:Chelating Agents; D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D014966:Xanthenes; C052455:nitr 5; C059715:Fluo-3; D008345:Manganese; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D015032:Zinc; D004072:Digitonin; D002118:Calcium; D016257:Fura-2