Fura-2 has become the most popular fluorescent probe with which to monitor dynarnic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2 AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2 AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accorate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4°C pre-chill to prevent intracellular organelle sequestration; (c) periormance of experiments at lower than physiological temperatures (i.e. 15-33°C) and use of ratio quantitation to remedy inaccuracles caused by dye leakage; (d) addition of N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calclum concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobieaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium. By accurately assessing the contribution of each of these potential artifacts, it is possible to use Fura-2 to accurately estimate cytosolic free calcium levels in intact living cells.
Bibliographical noteFunding Information:
nm excitatiowna velengtdhes pendhse avilyo n small can drastically altert he interpretatioofn the data. (in absoluttee rmsd) ecreaseosf the3 80n m values. However,v ariousp rotocolse xist to circumvent Such fluorescencise c loset o backgrounadn d hence thesep roblemsa nd allow the successfuul se of potentiallys,u bjectt o considerableex perimentaFl ura-in biologicrael search. variation. Use of the 365 run wavelengtahl so allowse stimateosf probep hotobleachianngd the quantitatioonf dye leakafgroem c ellsf romt hes ame Acknowledgements measurement. A potentialp roblemi n performingin situ We wish to thank Dr Gregory I. Gores for performing calibrationiss thatt he ionophoreuss edt o clamp using selective detergente xtraction. experiments quantifying the inea~ellular localization of Fura- calcium( i.e. ionomycin)m ay be inadequatteo This work was supported, in part, by Grants AGO7218 end equilibratcea lciuma crosst he plasmam embraneD K30874 from the National Institutes of Health, and the when concentratoiofn csa lcium obno ths ideso f the Gustavas and Louise PfeilTerF oundation membranaer em icromola(or rl ess).T hism ay result in nonequalc alciumc oncentrationasc rosst he membrane.H igherc oncentrationofs ionophote References mightb e employedb, ut we havee xperimentally observeuds ingd igitizedv ideom icroscopyth, at,in 1. hepatocyteasn d neonatacla rdiacm yocytesh, igh concentratioonfs i onomycinle adt o rapidl oss of quantitatibclyet osolic leveolfs F ura-fluorescence at ionomycin concentrationws hich did not equilibrate intarnad e xtracellulcaar lcium( Herman andL emasterus,n publishedda ta). In monolayers 4. and singlec ell work, one cannot usteh e lysis methodto getR maxa st hed yel eaksf romt hef ield of view. In vitro calibrationh as an additional advantagthea tt hec alibratiosno lutionc anb emade up to represenatn y pertinepnhty sicaclh aracteristics6. the investigatowri shest o duplicatefo r the cell’s internael nvironmentIt. alsoo bviates thpero blem of knowingw hethetrr uee quilibratioonf intraa nd extracellulacra lciumh as occurreda ftertreating cellsw ith ionomycoinr A23187H. oweverb,e cause it may be difficult to reproducet ruly the cytoplasmiec nvironmenct,a lciumc oncentrations calculatefrdo mi n vitroc alibratiocnu rvems ays how systematic errors.