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. 1987 Nov 1;105(5):2145–2155. doi: 10.1083/jcb.105.5.2145

Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells

PMCID: PMC2114834  PMID: 3680375

Abstract

The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+- free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.

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Selected References

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  1. Almers W., Neher E. The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading. FEBS Lett. 1985 Nov 11;192(1):13–18. doi: 10.1016/0014-5793(85)80033-8. [DOI] [PubMed] [Google Scholar]
  2. Baker P. F., Knight D. E. Exocytosis: control by calcium and other factors. Br Med Bull. 1986 Oct;42(4):399–404. doi: 10.1093/oxfordjournals.bmb.a072158. [DOI] [PubMed] [Google Scholar]
  3. Borgese N., Pietrini G. Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay. Biochem J. 1986 Oct 15;239(2):393–403. doi: 10.1042/bj2390393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Connor J. A. Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6179–6183. doi: 10.1073/pnas.83.16.6179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Di Virgilio F., Milani D., Leon A., Meldolesi J., Pozzan T. Voltage-dependent activation and inactivation of calcium channels in PC12 cells. Correlation with neurotransmitter release. J Biol Chem. 1987 Jul 5;262(19):9189–9195. [PubMed] [Google Scholar]
  6. Di Virgilio F., Pozzan T., Wollheim C. B., Vicentini L. M., Meldolesi J. Tumor promoter phorbol myristate acetate inhibits Ca2+ influx through voltage-gated Ca2+ channels in two secretory cell lines, PC12 and RINm5F. J Biol Chem. 1986 Jan 5;261(1):32–35. [PubMed] [Google Scholar]
  7. Dunn W. A., Hubbard A. L., Aronson N. N., Jr Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J Biol Chem. 1980 Jun 25;255(12):5971–5978. [PubMed] [Google Scholar]
  8. Goldstein J. L., Brown M. S. Binding and degradation of low density lipoproteins by cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem. 1974 Aug 25;249(16):5153–5162. [PubMed] [Google Scholar]
  9. Goligorsky M. S., Hruska K. A., Loftus D. J., Elson E. L. Alpha 1-adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: evidence for compartmentalization of quin-2 and fura-2. J Cell Physiol. 1986 Sep;128(3):466–474. doi: 10.1002/jcp.1041280316. [DOI] [PubMed] [Google Scholar]
  10. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  11. Highsmith S., Bloebaum P., Snowdowne K. W. Sarcoplasmic reticulum interacts with the Ca(2+) indicator precursor fura-2-am. Biochem Biophys Res Commun. 1986 Aug 14;138(3):1153–1162. doi: 10.1016/s0006-291x(86)80403-x. [DOI] [PubMed] [Google Scholar]
  12. Jackson T. R., Hallam T. J., Downes C. P., Hanley M. R. Receptor coupled events in bradykinin action: rapid production of inositol phosphates and regulation of cytosolic free Ca2+ in a neural cell line. EMBO J. 1987 Jan;6(1):49–54. doi: 10.1002/j.1460-2075.1987.tb04717.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Keith C. H., Ratan R., Maxfield F. R., Bajer A., Shelanski M. L. Local cytoplasmic calcium gradients in living mitotic cells. 1985 Aug 29-Sep 4Nature. 316(6031):848–850. doi: 10.1038/316848a0. [DOI] [PubMed] [Google Scholar]
  14. Martinez A. O., Vigil A., Vila J. C. Flow-cytometric analysis of mitochondria-associated fluorescence in young and old human fibroblasts. Exp Cell Res. 1986 Jun;164(2):551–555. doi: 10.1016/0014-4827(86)90053-4. [DOI] [PubMed] [Google Scholar]
  15. Neher E., Almers W. Fast calcium transients in rat peritoneal mast cells are not sufficient to trigger exocytosis. EMBO J. 1986 Jan;5(1):51–53. doi: 10.1002/j.1460-2075.1986.tb04176.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nemeth E. F., Wallace J., Scarpa A. Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+. J Biol Chem. 1986 Feb 25;261(6):2668–2674. [PubMed] [Google Scholar]
  17. Pandiella-Alonso A., Malgaroli A., Vicentini L. M., Meldolesi J. Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells. FEBS Lett. 1986 Nov 10;208(1):48–51. doi: 10.1016/0014-5793(86)81529-0. [DOI] [PubMed] [Google Scholar]
  18. Pandiella A., Malgaroli A., Meldolesi J., Vicentini L. M. EGF raises cytosolic Ca2+ in A431 and Swiss 3T3 cells by a dual mechanism. Redistribution from intracellular stores and stimulated influx. Exp Cell Res. 1987 May;170(1):175–185. doi: 10.1016/0014-4827(87)90127-3. [DOI] [PubMed] [Google Scholar]
  19. Poenie M., Alderton J., Steinhardt R., Tsien R. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science. 1986 Aug 22;233(4766):886–889. doi: 10.1126/science.3755550. [DOI] [PubMed] [Google Scholar]
  20. Poenie M., Alderton J., Tsien R. Y., Steinhardt R. A. Changes of free calcium levels with stages of the cell division cycle. Nature. 1985 May 9;315(6015):147–149. doi: 10.1038/315147a0. [DOI] [PubMed] [Google Scholar]
  21. Pritchard K., Ashley C. C. Na+/Ca2+ exchange in isolated smooth muscle cells demonstrated by the fluorescent calcium indicator fura-2. FEBS Lett. 1986 Jan 20;195(1-2):23–27. doi: 10.1016/0014-5793(86)80122-3. [DOI] [PubMed] [Google Scholar]
  22. Rasmussen H. The calcium messenger system (2). N Engl J Med. 1986 May 1;314(18):1164–1170. doi: 10.1056/NEJM198605013141807. [DOI] [PubMed] [Google Scholar]
  23. Swennen L., Baes M., Schramme C., Denef C. Beta-adrenergic stimulation of adenosine-3',5'-monophosphate (c-AMP) in primary cultures of rat anterior pituitary cell populations separated by unit gravity sedimentation. Relationship to growth hormone and prolactin release and to nonsecreting cells. Neuroendocrinology. 1985 Jan;40(1):78–83. doi: 10.1159/000124055. [DOI] [PubMed] [Google Scholar]
  24. Tsien R. Y. New tetracarboxylate chelators for fluorescence measurement and photochemical manipulation of cytosolic free calcium concentrations. Soc Gen Physiol Ser. 1986;40:327–345. [PubMed] [Google Scholar]
  25. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tsien R. Y., Rink T. J., Poenie M. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium. 1985 Apr;6(1-2):145–157. doi: 10.1016/0143-4160(85)90041-7. [DOI] [PubMed] [Google Scholar]
  27. Wanke E., Ferroni A., Malgaroli A., Ambrosini A., Pozzan T., Meldolesi J. Activation of a muscarinic receptor selectively inhibits a rapidly inactivated Ca2+ current in rat sympathetic neurons. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4313–4317. doi: 10.1073/pnas.84.12.4313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Williams D. A., Fogarty K. E., Tsien R. Y., Fay F. S. Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2. Nature. 1985 Dec 12;318(6046):558–561. doi: 10.1038/318558a0. [DOI] [PubMed] [Google Scholar]

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