Calcium Signaling Protocols Edited by David G. Lambert Methods in Molecular Biology Methods in Molecular Biology TM TM VOLUME 114 HUMANA PRESS HUMANA PRESS Calcium Signaling Protocols Edited by David G. Lambert Fluorescent Measurement of [Ca 2+ ] c 3 1 3 From: Methods in Molecular Biology, Vol. 114: Calcium Signaling Protocols Edited by: D. G. Lambert © Humana Press Inc., Totowa, NJ Fluorescent Measurement of [Ca 2+ ] c Basic Practical Considerations Alec W. M. Simpson 1. Introduction It is extremely difficult to write a prescriptive account of how to measure cytosolic-free Ca 2+ ([Ca 2+ ] c ) that will suit all potential investigators, given the wide diversity of fluorescent Ca 2+ indicators that are now available, the variety of cells to be investigated, and an increasing range of detection equipment that can be used. Therefore, this chapter is designed to provide the user with suffi- cient background in the technology so that he or she can move toward develop- ing a protocol that will suit the cells, the experimental objectives, and the equipment available to the investigator. The main approaches to measuring [Ca 2+ ] c before the synthesis of fluores- cent Ca 2+ indicators involved using the Ca 2+ -activated photoprotein aequorin, Ca 2+ -selective microelectrodes, or absorbance indicators (1). The use of aequorin and microelectrodes was generally restricted to large cells (usually from invertebrates) that were easy to handle and manipulate with micropipets. With a few notable exceptions (e.g., injection of hepatocytes and myocytes with aequorin by Cobbold and colleagues [2,3]), these approaches were not applied to the wide diversity of cells present in mammalian tissues. The use of absorbance dyes did not become widespread since they are not very sensitive to the typical [Ca 2+ ] c found in cells, and did not offer any real potential for investigating [Ca 2+ ] c in monolayers or single cells. The synthesis of quin2 by Tsien (4,5) in the early 1980s heralded a new era in the measurement of Ca 2+ , by making available fluorescent probes that could be readily introduced into living cells. The most commonly used fluorescent Ca 2+ indicator at present is fura-2, which, along with indo-1, 4Simpson formed a new generation of ratiometric indicators also designed by Tsien and colleagues (6). The Ca 2+ -binding properties of these indicators is formed by the presence of a tetracarboxylic acid core as found in the Ca 2+ -chelator EGTA. Whereas the Ca 2+ binding of EGTA is highly pH dependent, the original Ca 2+ indicator quin2 and its successors were designed around an EGTA derivative, BAPTA, also synthesized by Tsien (7). For a compound to act as an intracellular Ca 2+ indicator, selectivity of the indicator for Ca 2+ over other physiologically important ions is essential. EGTA already showed a much greater selectivity for Ca 2+ over Mg 2+ , Na + and K + , but unfortunately, its Ca 2+ binding is very pH sensitive. Cells undergo physiological changes in pH (8), which in the case of an EGTA-like chelator would affect the reported [Ca 2+ ]. Calibrating a pH-sen- sitive Ca 2+ indicator would be quite difficult, since small changes in pH of the calibration solutions would affect the measured fluorescence and the K d for Ca 2+ . The synthesis of BAPTA, a largely pH-insensitive Ca 2+ chelator, was therefore an important step in the development of fluorescent probes for mea- suring [Ca 2+ ] c (7). Since the introduction of quin2, fura-2, and indo-1, numerous other fluores- cent Ca 2+ indicators have been synthesized, each with varying fluorescence characteristics and K d s for Ca 2+ see ref. 9; Tables 1–3). The fundamental prop- erties of these indicators are similar, in that the binding of Ca 2+ produces a wavelength shift in either the excitation or emission fluorescence spectra (6,9). When there is little or no shift in the excitation spectra, a Ca 2+ -dependent change in the emission intensity is used to report changes in Ca 2+ (5,9). This can arise from Ca 2+ -dependent changes in the intensity of absorbance or quan- tum efficiency. In terms of fluorescence properties, the indicators can be divided into two main groups, those that are excited by near ultraviolet (UV) wavelengths 340–380 nm (e.g., quin2, fura-2, and indo-1) and those that are excited with visible light at or above 450 nm (e.g., fluo-3, Calcium Green, rhod-2; see refs. 9 and 10) The fluorophores for the visible indicators tend to be fluoroscein and rhodamine derivatives. This is advantageous since a great deal of fluo- rescence instrumentation has been designed for use with fluoroscein- and rhodamine-based dyes. 2. Overview 2.1. Single Excitation Indicators The first of this family is quin2, Tsien’s (5) original fluorescent Ca 2+ indica- tor. When excited at 340 nm, an increase in emission intensity peaking at 505 nm is observed on binding Ca 2+ . Under physiological conditions, quin2 has a K d of Fluorescent Measurement of [Ca 2+ ] c 5 115 nM, making it useful for measuring [Ca 2+ ] c changes at or close to those found in unstimulated (resting) cells; however, the dye is of little use in moni- toring changes in [Ca 2+ ] c in excess of 1 µM. Poor quantum efficiency has limited the use of this indicator, especially after the introduction of the more fluorescent ratiometric probes. However, quin2 does have some useful proper- ties; like BAPTA, it is a very good buffer of [Ca 2+ ] c , and its use has allowed Ca 2+ -independent phenomena to be observed (11,12). Subsequently improved single excitation indicators have been developed that are more fluorescent and have K d s for Ca 2+ between ~200 nM and 20 µM (9,10,13) (see Table 1). These indicators include fluo-3 (10), and the Calcium Green and Calcium Orange series of indicators. With these indicators there is little or no shift in either the excitation or emission spectra; however, a marked increase in fluorescence intensity can be observed on Ca 2+ binding. Calcium Green-2™ has a K d of 550 nM (Table 1) and produces approx 100-fold increase in fluorescence between being Ca 2+ -free and Ca 2+ -saturated. For fluo-3 this increase is reported to be approx 200-fold. The fluo and Calcium Green indicators all have peak excitation spectra at or close to 490 nm (see Table 1), allowing them to be readily used with argon-ion lasers (488 nm excitation). Peak emission lies close to 530 nm. There are Ca 2+ indicators that can be excited even at longer wavelengths, e.g., rhod-2, the Calcium Crimson and Calcium Orange series, and KJM-1 (Table 1). Rhod-2 is excited at 520 nm, with a peak emission at 580 nm (10), and has been used to measure mitochondrial Ca 2+ rather than [Ca 2+ ] c (14). Fura-Red (strictly a ratiometric indicator) when excited at wave- lengths close to 480 nm can be used in combination with fluo-3 to obtain a ratio derived from their respective 530- and 650-nm emission signals. Thus, combinations of visible excitation indicators can be used to obtain ratio mea- sures of [Ca 2+ ] c (15,16). 2.1.1. Visible Excitation Indicators Visible wavelength indicators are attractive because they can avoid problems such as light absorbance by optical elements and cellular autofluorescence. The lower excitation energies of the longer wavelengths also means that photobleaching is reduced. The visibly excited dyes are more suited to the laser- based illumination systems used in confocal microscopy and flow cytometry. The advantage of having a range of indicators that can be excited at different wavelengths is that combinations of ion-indicators can be used together. Thus, Ca 2+ can be monitored simultaneously with other physiologically important ions such as Na + or H + (17–19). Moreover, Ca 2+ can be monitored using indi- cators in separate domains as with simultaneous measurements of intracellular and extracellular Ca 2+ (20). 6Simpson Table 1 Single Excitation Wavelength Indicators AM Absorbance Emission Indicator Source a UV/V K d loading –Ca 2+ +Ca 2+ –Ca 2+ +Ca 2+ Comments Quin2 MP/TL UV 60 b (115) d ✔ 352 332 492 498 High intracellular buffering Methoxyquin2MF MP UV 65 b ✔ 352 332 492 498 MethoxyquinMF 19 for nuclear magnetic resonance Oregon Green 488 MP V 170 b ✔ 494 494 523 523 Designed for argon-ion lasers BAPTA-1 Calcium Orange™ MP V 185 b (380) e ✔ 549 549 575 576 See ref. 13 Calcium Crimson™ MP V 185 b (221) c ✔ 590 589 615 615 See ref. 13 Calcium Green-1™ MP V 190 b (221) e ✔ 503 506 534 533 See ref. 13; fluorescence lifetime measurements Calcium Green C 18 MP V 280 b (62) f ✘ 509 509 530 530 Near membrane Ca 2+ indicator; K d affected by lipids; ref. 53 Fluo-3 MP/TL V 390 b ✔ 506 506 526 526 AM ester and Ca 2+ -free forms only weakly fluorescent; large increase in fluorescence on Ca 2+ binding KJM-1 TL V 500 c ✔ 560 560 Em640 Calcium Green-2™ MP V 550 ✔ 503 503 536 536 Fluo LR TL V 550 c ✔ 506 506 525 525 Leakage-resistant indicator Rhod-2 MP/TL V 570 b ✔ 549 552 571 571 Can be loaded as dihydro derivative; will locate in mitochondria and peroxisomes Oregon Green 488 MP V 580 b ✔ 494 494 523 523 Designed for argon-ion lasers BAPTA-2 Fluorescent Measurement of [Ca 2+ ] c 7 Fluo-535 TL V 800 ✔ 535 535 560 560 Lower-affinity derivative (Fluo-535FF) also available Magnesium Green™ MP V 6 µM b ✔ 506 506 531 531 K d in 0-Mg 2+ ; indicator will be Mg 2+ sensitive Calcium Green-5N™ MP V 14 µM b ✔ 506 506 532 532 Calcium Orange-5N™ MP V 20 µM b ✔ 549 549 582 582 Exhibits very fast kinetics suitable for millisecond time resolution; ref. 77 Oregon Green 488 MP V 20 µM b ✔ 494 494 521 521 Designed for argon-ion lasers BAPTA-5N™ Fluo-3FF TL V 41 µM b ✔ Ex 515 526 526 a MP, Molecular Probes; TL, Teflabs; MP/TL, Molecular Probes, Teflabs, and other suppliers. b K d determined in 100 mM KCl, pH 7.2, at 22°C. c K d determined at pH 7.2 and 22°C. d K d determined in 100 mM KCl, pH 7.05, at 37°C. e Values taken from ref. 13. f K d reported to be 230 nM at 0.1M ionic strength, pH 7.2, at 22°C and 62 nM in the presence of phospholipid vesicles. See ref. 50. 8Simpson Table 2 Dual Excitation Ratiometric Indicators AM Absorbance Emission Indicator Source a UV/V K d loading –Ca 2+ +Ca 2+ –Ca 2+ +Ca 2+ Comments Fura Red™ MP V 140 b ✔ 472 436 657 637 Low quantum yield; used in combination with single ex indicators to obtain ratios Fura-2 MP/TL UV 145 b (224) c ✔ 363 335 512 505 Fura C 18 MP UV 150 b ✘ 365 338 501 494 Lipophilic near-membrane Ca 2+ indicator Fura PE3 TL UV 250 d ✔ 364 335 508 500 Leakage-resistant indicator FFP18 TL UV 400 ✔ 364 335 502 495 Lipophilic near-membrane Ca 2+ indicator Bis-fura-2 MP UV 370 b (525) e ✘ 366 338 511 505 Brighter and has lower affinity than fura-2; not available as an ester BTC MP V 7 µM b ✔ 464 401 533 529 Visible-excitation ratiometric indicator Mag-fura-2 MP UV 25 µM b ✔ 369 329 511 508 K d in 0-Mg 2+ ; indicator will be (Furaptra) Mg 2+ sensitive Mag-fura-5 MP UV 28 µM b ✔ 369 330 505 500 K d in 0-Mg 2+ ; indicator will be Mg 2+ sensitive Fura-2FF TL UV 35 µM d ✔ 335 364 512 505 a MP, Molecular Probes; TL, Teflabs; MP/TL, Molecular Probes, Teflabs, and other suppliers. b K d determined in 100 mM KCl, pH 7.2, at 22°C. c K d determined in 100 mM KCl, pH 7.05, at 37°C. d Conditions for K d determined not defined. e Conditions same as in footnote b, but with 1 mM Mg 2+ present. 8 Fluorescent Measurement of [Ca 2+ ] c 9 Table 3 Dual Emission Ratiometric Indicators AM Absorbance Emission Indicator Source a UV/V K d loading –Ca 2+ +Ca 2+ –Ca 2+ +Ca 2+ Comments Indo-1 MP/TL UV 230 b (250) c ✔ 346 330 475 401 Indo PE3 TL UV 260 d ✔ 346 330 475 408 Leakage-resistant indicator FIP18 TL UV 450 d ✔ 346 330 475 408 Lipophilic near-membrane Ca 2+ indicator Indo-1FF TL UV 33 µM d ✔ Ex 348 475 408 Mag-indo-1 MP UV 35 µM e ✔ 349 328 480 390 K d in 0-Mg 2+ ; indicator will be Mg 2+ sensitive a MP, Molecular Probes; TL, Teflabs; MP/TL, Molecular Probes, Teflabs, and other suppliers. b K d determined in 100 mM KCl, pH 7.2, at 22°C. c K d determined in 115 mM KCl, 20 mM NaCl, 10 mM K-MOPS, pH 7.05, 1 mM Mg 2+ at 37°C. d Conditions for K d determination not defined. e K d determined in 100 mM KCl, 40 mM HEPES, pH 7.0, at 22°C. 9 10 Simpson 2.1.2. Caged Compounds Bioactive molecules can be incorporated into physiologically inert (caged) molecules and subsequently released in a controlled manner by photolysis of the chemical “cage.” Introduction of the visible excitation indicators has allowed [Ca 2+ ] c to be measured during UV-induced flash photolysis of caged compounds such as caged Ins(1,4,5)P 3 and Nitr-5 (caged Ca 2+ ) (21). This advance has enabled second messengers to be manipulated in a controlled man- ner while simultaneously monitoring [Ca 2+ ] c . 2.2. Dual Excitation Indicators Fura-2 is the archetypal dual excitation Ca 2+ indicator (6). In low Ca 2+ , fura-2 shows a broad excitation spectrum between 300 and 400 nm, with a peak at approx 370 nm. On Ca 2+ binding, the excitation peak increases in intensity and also shifts further into the UV (Fig. 1). Consequently, if the dye is excited at 340 nm (emission monitored at 510 nm), Ca 2+ binding will produce an increase in fluorescence, whereas a decrease in the fluorescent signal is observed when the dye is excited at 380 nm (Figs. 1 and 2). When the dye is excited in quick Fig. 1. Ca 2+ -free and Ca 2+ -saturated excitation spectra of fura-2. The two spectra coincide at 360 nm, the isobestic (or isoemissive) point. It can be seen that when Ca 2+ binds, the fluorescence signal will increase when the indicator is excited at 340 nm, remain the same when it is excited at 360 nm, and decrease when it is excited at 380 nm. Fluorescent Measurement of [Ca 2+ ] c 11 succession at 340 and 380 nm, a ratio of the respective emission signals can be used to monitor [Ca 2+ ]. Ratiometric measurements have a number of advan- tages over single wavelength probes. The ratio signal is not dependent on dye concentration, illumination intensity, or optical path length. Therefore, spatial variations in these parameters will not affect the estimations of [Ca 2+ ] c . Such factors are especially important if the dyes are to be used for imaging of [Ca 2+ ] c , which illumination intensity and optical properties vary across the field of view (6,22). Dye leakage and photobleaching frequently lead to a loss of indicator during an experiment; thus, the active indicator concentration cannot be assumed to be constant (23,24). Under such conditions, a ratiometric indicator gives a more stable measure of [Ca 2+ ] c than could be obtained from a single excitation indicator. Ratiometric measurements also produce an additional increase in sensitivity. A further useful property of ratiometric indicators is the presence of an isobestic or isoemissive point. For example, when fura-2 is excited at 360 nm, Fig. 2. The typical signals obtained from a fura-2–loaded cell when it is excited alternately at 340 and 380 nm. Agonist stimulation will cause an increase in the 340-nm signal and a decrease in the 380-nm signal. Addition of a Ca 2+ ionophore (Iono), in the presence of Ca 2+ , will give F 340max and F 380min , whereas subsequent addition of EGTA will give F 380max and F 340min . The time taken to reach F 340max and F 380min after addition of ionophore and EGTA can vary and be in excess of 30 min. Curve-fitting the decay toward R min has been suggested as a strategy to speed up the calibration process (34). The long time period required to obtain R min (340min/380max) is not ideal for imaging experiments since the dimensions of the cell may change during the calibration. 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Pozzan, T., and Rink, T J (1982) Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new intracellularly trapped fluorescent indicator J Cell Biol 94, 325–334 6 Grynkiewicz, G., Poenie, M., and Tsien, R Y (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties J Biol Chem 260, 3440–3450 7 Tsien, R Y (1980) New calcium indicators and buffers... in Chapter 1, fura-2–free acid is Ca2+ sensitive but membrane impermeant To effect cell loading, cells are incubated with fura-2 pentaacetoxymethyl From: Methods in Molecular Biology, Vol 114: Calcium Signaling Protocols Edited by: D G Lambert © Humana Press Inc., Totowa, NJ 31 32 Hirst et al (AM) ester; this form of the dye is Ca2+ insensitive Once inside the cell, esterase enzymes sequentially cleave . Calcium Signaling Protocols Edited by David G. Lambert Methods in Molecular Biology Methods in Molecular Biology TM TM VOLUME 114 HUMANA PRESS HUMANA PRESS Calcium Signaling Protocols Edited. Lambert Fluorescent Measurement of [Ca 2+ ] c 3 1 3 From: Methods in Molecular Biology, Vol. 114: Calcium Signaling Protocols Edited by: D. G. Lambert © Humana Press Inc., Totowa, NJ Fluorescent Measurement. for argon-ion lasers BAPTA-1 Calcium Orange™ MP V 185 b (380) e ✔ 549 549 575 576 See ref. 13 Calcium Crimson™ MP V 185 b (221) c ✔ 590 589 615 615 See ref. 13 Calcium Green-1™ MP V 190 b (221) e ✔