Visible light–excitable Ca2+ indicators offer several advantages over UV light–excitable indicators:
- Efficient excitation with most laser-based instrumentation, including confocal laser-scanning microscopes and flow cytometers
- Stronger absorption by the dyes, which permits the use of lower dye concentrations and therefore lower phototoxicity to live cells
- Large Ca2+-dependent fluorescence intensity increases, resulting in sensitive detection of Ca2+ transients
- Reduced interference from sample autofluorescence and light scatter
- Compatibility with photoactivatable ("caged") probes and other UV light–absorbing reagents, increasing options for multiparameter measurements
The Ca2+ indicator fluo-3 (Figure 19.3.1) was developed by Tsien and colleagues for use with visible-light excitation sources in flow cytometry and confocal laser-scanning microscopy. More recently, fluo-3 imaging has been extended to include two-photon excitation techniques () and total internal reflection fluorescence (TIRF) microscopy. Fluo-3 imaging has revealed the spatial dynamics of many elementary processes in Ca2+ signaling (, ). Since about 1996, fluo-3 has also been extensively used in cell-based high-throughput screening assays for drug discovery. Fluo-3 is essentially nonfluorescent unless bound to Ca2+ and exhibits a quantum yield at saturating Ca2+ of ~0.14 (Figure 19.3.2) and a Kd for Ca2+ of 390 nM (measured at 22°C using our Calcium Calibration Buffer Kits). The intact acetoxymethyl (AM) ester derivative of fluo-3 is almost nonfluorescent, unlike the AM esters of fura-2 and indo-1. The green-fluorescent emission (~525 nm) of Ca2+-bound fluo-3 is conventionally detected using optical filter sets designed for fluorescein (FITC).
In a careful study of the spectral properties of highly purified fluo-3, Harkins, Kurebayashi and Baylor characterized the effects of pH and viscosity on Ca2+ measurements with fluo-3 and demonstrated that binding of the indicator to proteins has a significant effect on its Kd for Ca2+ (Comparison of in vitro and in situ Kd values for various Ca2+ indicators—Table 19.2). The temperature dependence of the Kd for fluo-3 has also been reported. In addition, the fluorescence output of fluo-3—the product of the molar absorptivity and the fluorescence quantum yield—may also vary significantly in different cellular environments.
Fluo-3 exhibits an at least 100-fold Ca2+-dependent fluorescence enhancement. However, fluo-3 lacks a significant shift in emission or excitation wavelength upon binding to Ca2+, which precludes the use of ratiometric measurements (Figure 19.3.3). Simultaneous loading of cells with fluo-3 and our Invitrogen™ Fura Red™ indicator (see below), which exhibit reciprocal shifts in fluorescence intensity upon binding Ca2+, has enabled researchers to make ratiometric measurements of intracellular Ca2+ (Figure 19.3.4) using confocal laser-scanning microscopy () or flow cytometry. For ratiometric measurements, fluo-3 (or fluo-4) can also be co-loaded into cells with a spectrally distinct Ca2+-insensitive dye, such as Invitrogen™ CellTracker™ Orange CMRA (C34551, Membrane-Permeant Reactive Tracers—Section 14.2) or Invitrogen™ CellTrace™ Calcein Red-Orange, AM (C34851, Viability and Cytotoxicity Assay Reagents—Section 15.2). It is common practice when loading neurons in brain slices via patch pipette infusion with green-fluorescent calcium indicators, such as fluo-3, fluo-4, fluo-5F and Invitrogen™ Oregon Green™ 488 BAPTA-1, to add in a Ca2+-insensitive structural marker such as Invitrogen™ Alexa Fluor™ 594 hydrazide (A10438, A10442; Polar Tracers—Section 14.3).
Fluo-3 is available as a cell-impermeant potassium salt (F3715) or ammonium salt (F1240). The cell-permeant fluo-3 AM is available as a 1 mg vial (F1241), as a set of 20 vials each containing 50 µg (F1242), as a set of 10 vials each containing 50 µg of our high-purity grade fluo-3 AM (F23915) and as a 1 mM solution in DMSO (F14218). A set of 40 vials, each containing 1 mg of fluo-3 AM, is available at a discounted price to provide a larger quantity for high-throughput screening applications (F14242).
Figure 19.3.1 Fluo indicators.
Figure 19.3.2 Comparison of fluorescence intensity responses to Ca2+ for the fluo-3 (F1240, F3715) and Calcium Green-1 (C3010MP) indicators. Responses were calculated from the Ca2+ dissociation constants for the two indicators and the extinction coefficients and fluorescence quantum yields of their ion-free and ion-bound forms. They, therefore, represent the relative fluorescence intensities that would be obtained from equal concentrations of the two indicators excited and detected at their peak wavelengths.
Fluo-4 (), an analog of fluo-3 with the two chlorine substituents replaced by fluorine atoms (Figure 19.3.1), exhibits a Kd for Ca2+ of 345 nM (measured at 22°C using our Calcium Calibration Buffer Kits); the temperature dependence of the Kd for fluo-4 has been reported. The fluorescence quantum yields of Ca2+-bound fluo-3 and fluo-4 are essentially identical. Significantly, however, the absorption maximum of fluo-4 is blue-shifted about 12 nm, as compared with fluo-3, resulting in increased fluorescence excitation at 488 nm and consequently higher signal levels for confocal laser-scanning microscopy (Figure 19.3.5), flow cytometry and microplate screening applications (Figure 19.3.6, Figure 19.3.7).
Intracellular Ca2+ measurements using fluo-3 and fluo-4 have become essential for certain types of high-throughput pharmacological screening. Applications of this technology include screening for compounds that activate or deactivate G-protein–coupled receptors and identifying receptors for ligands known to be pharmacologically active. Molecular Devices FLIPR (Fluorometric Imaging Plate Reader) system has been the leading instrument platform for these measurements (Figure 19.3.7). Parallel comparisons of fluo-4 with fluo-3, carried out in collaboration with researchers at Molecular Devices, show that the advantages of fluo-4 in microscopy and solution fluorometry measurements are replicated in the FLIPR system. For example, fluo-4 generates the same fluorescence response as fluo-3 to carbachol-stimulated Ca2+ activation in Chinese hamster ovary (CHO) cells using half the AM ester–loading concentration and half the incubation time (Parallel performance comparison of fluo-3 and fluo-4 on Molecular Devices FLIPR system—Table 19.3). When fluo-4 is substituted for fluo-3 (i.e., using identical loading protocols), fluorescence signals are at least doubled. The stronger fluorescence signals provided by fluo-4 are particularly advantageous in cell types such as human embryonic kidney (HEK 293) cells, which are seeded at low densities for pharmacological screening assays.
Fluo-4 is available as a cell-impermeant potassium salt (F14200) or as its cell-permeant AM ester. The AM ester is available specially packaged as a 1 mM solution in DMSO (F14217), as a set of 10 vials, each containing 50 µg (F14201, F23917) or—for high-throughput screening applications—as a set of five vials, each containing 1 mg (F14202). We also offer both low-affinity and high-affinity 10,000 MW dextran conjugates of the fluo-4 indicator (F14240, F36250; Fluorescent Ca2+ Indicator Conjugates—Section 19.4).
We also offer the Fluo-4 Direct Calcium Assay Kits (F10471, F10472, F10473) which offer a proprietary assay formulation that allows direct addition to wells containing cells growing in culture media without the requirement of media removal or a wash step. Eliminating the media removal step from the workflow can result in lower variability and higher Z´ values compared with the standard fluo-4 assay, while also providing an easier and faster assay. Contributions to baseline fluorescence by the growth medium are eliminated by the addition of a suppression dye, which reduces background fluorescence. Another source of background fluorescence is extrusion of the indicator out of the cell by organic anion transporters. To address this background issue, the Fluo-4 Direct Calcium Assay Kits provide a proprietary, water-soluble probenecid, which inhibits this transport and reduces the baseline signal. The water-soluble form of probenecid is easy to dissolve in physiological buffers, unlike the conventionally used free acid form, which must be initially dissolved in strong base.
Each Fluo-4 Direct Calcium Assay Kit provides:
- Fluo-4 calcium assay reagent
- Water-soluble probenecid
- Fluo-4 Direct calcium assay buffer (only provided with the Starter pack F10471 and the Surveyor pack F10472)
- Detailed protocols (Fluo-4 Direct Calcium Assay Kits)
Sufficient material is supplied for 20 × 96- or 384-well plates (Starter pack, F10471), 20 × 96- or 384-well plates (Surveyor pack, F10472) or 200 × 96- or 384-well plates (High-throughput pack, F10473). The Fluo-4 Direct Calcium Assay Kits are designed for microplates and high-throughput screening (HTS), and the assay can be performed on adherent as well as nonadherent cells. Water-soluble probenecid is also available separately (P36400, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8).
The Fluo-4 NW Calcium Assay Kits (F36205, F36206) provide a proprietary assay formulation that requires neither a wash step nor a quencher dye. The fluo-4 NW indicator is nonfluorescent and stable in pH 7–7.5 buffer for several hours, so spontaneous conversion to the Ca2+-sensitive form is not a significant source of background fluorescence. Contributions to baseline fluorescence by the growth medium (e.g., esterase activity, proteins interacting with receptors of interest, or phenol red) are eliminated by removing the medium prior to adding the indicator dye to the wells. A water-soluble form of probenecid is included to inhibit extrusion of the indicator out of the cell by organic anion transporters.
Each Fluo-4 NW Calcium Assay Kit provides:
- Fluo-4 NW dye mix
- Water-soluble probenecid
- Assay buffer (only provided in the Fluo-4 NW Calcium Assay Kit starter pack with buffer F36206)
- Detailed protocols (Fluo-4 NW Calcium Assay Kits)
The Fluo-4 NW Calcium Assay Kit starter pack with buffer (F36206) contains enough materials for 10 microplates and includes assay buffer; the Fluo-4 NW Calcium Assay Kit for high-throughput assays (F36205) contains enough materials for 100 microplates and does not include the assay buffer. These kits are designed for microplates and HTS, and the assay can be performed on adherent as well as nonadherent cells.
Figure 19.3.5 Fast confocal recording of spontaneous Ca2+ sparks in a rat ventricular myocyte. Single myocytes were isolated from rat hearts and loaded with fluo-4 AM (F14201, F14202, F14217, F23917). Images of loaded cells were obtained with a PerkinElmer UltraVIEW LCI confocal imaging system at a constant frame rate of 66 frames/second. Panel A illustrates the outline of a single cell and the position of the four regions of interest that were averaged to produce the kinetic traces shown in panel B. The traces are color coded in accordance with the regions marked in panel A and show numerous spontaneous Ca2+ sparks. Panel C illustrates a sequence of confocal images taken around the black region in panel A, indicating the spatiotemporal properties of an individual Ca2+ spark. The image was contributed by Peter Lipp, The Babraham Institute, Cambridge, UK, and reproduced with permission from Biomedical Products (2001) 9:52.
Figure 19.3.7 High-throughput Ca2+ influx assays for G-protein–coupled receptor activation. CHO (DHFR-) cells, co-transfected with the orphanin FQ receptor and Gαqi3 chimeric G-protein, were loaded with 4 µM fluo-3 AM (F1241, F1242, F14218, F14242, F23915) or fluo-4 AM (F14201, F14202, F14217, F23917) for 60 minutes at 37°C. Ca2+-dependent fluorescence traces (1 data point/second, excitation at 488 nm) from loaded cell samples in a 96-well microplate were measured simultaneously by a Fluorometric Imaging Plate Reader (FLIPR). Ca2+ transients were initiated by addition of 25 nM orphanin FQ (nociceptin), as indicated by the baseline discontinuities at approximately 10 seconds. Each trace represents the average of five transient recordings from separate microplate wells. The data were supplied by Sven Merten, Hans-Peter Nothacker and Olivier Civelli, University of California, Irvine.
Rhod-2 and X-Rhod-1
The long-wavelength Ca2+ indicators rhod-2 (Figure 19.3.8, ) and X-rhod-1 are valuable for measuring Ca2+in cells and tissues that have high levels of autofluorescence and also for detecting Ca2+ release generated by photoreceptors and photoactivatable chelators. Our chemists have optimized the purification of rhod-2, yielding a highly purified preparation that shows greater than 100-fold enhancement in fluorescence upon binding Ca2+(Figure 19.3.9). The Kd for Ca2+ of rhod-2 in the absence of Mg2+ has been determined to be 570 nM (measured at 22°C using our Calcium Calibration Buffer Kits), which is considerably lower than that cited in the original paper on rhod-2. Rhod-2 is available as a cell-impermeant potassium salt (R14220) or as a cell-permeant AM ester in either a 1 mg vial (R1244) or specially packaged as a set of 20 vials, each containing 50 µg (R1245MP).
X-rhod-1 is a Ca2+ indicator with excitation/emission maxima of ~580/602 nm and a Kd for Ca2+ of 700 nM (measured at 22°C using our Calcium Calibration Buffer Kits). It has spectral characteristics that are similar to our Invitrogen™ Calcium Crimson™ indicator (see below), but the fluorescence response of X-rhod-1 is much more sensitive to Ca2+ binding (Figure 19.3.10). The long-wavelength emission characteristics of X-rhod-1 allow simultaneous detection Ca2+ transients and green-fluorescent protein (GFP) with minimal crosstalk (Using Organic Fluorescent Probes in Combination with GFP—Note 12.1). X-rhod-1 is available as a cell-permeant AM ester (X14210), specially packaged as a set of 10 vials, each containing 50 µg.
Figure 19.3.8 Rhod indicators with varying Ca2+ affinities.
Figure 19.3.9 Fluorescence emission spectra of rhod-2 (R14220) in solutions containing 0–39.8 µM free Ca2+. The spectrum for the Ca2+-free solution is indistinguishable from the baseline.
Figure 19.3.10 Fluorescence emission spectra of the tripotassium salt of X-rhod-1 in solutions containing 0—39.8 µM free Ca2+.
Rhod-3 Imaging Kit
As compared with other red-fluorescent calcium dyes such as rhod-2 AM, rhod-3 AM is an improved red-shifted calcium indicator that displays a more uniform cytosolic distribution and improved signal. The cationic nature of rhod-2 AM results in potential-driven, subcellular localization (). Imaging studies with the rhod-3 AM, however, show minimal subcellular localization. In the presence of Invitrogen™ PowerLoad™ concentrate and probenecid, the cell-permeant, nonfluorescent rhod-3 AM can be passively loaded into the cells, where intracellular esterases cleave the dye to the cell-impermeant, active form that fluoresces upon Ca2+ binding. Rhod-3 exhibits a large increase (>2.5 fold) in fluorescence upon binding Ca2+ and very low fluorescence in the absence of Ca2+ binding. Rhod-3 has a Kd of 570 nM for Ca2+ (determined at 22°C in 30 mM MOPS, pH 7.2 with 100 mM KCl).
We offer the Rhod-3 Imaging Kit (R10145), which provides:
- Rhod-3 AM
- Dimethylsulfoxide (DMSO)
- PowerLoad concentrate
- Water-soluble probenecid
- Detailed protocols (Rhod-3 Imaging Kit)
Sufficient reagents are supplied for 10 assays using the protocol provided. PowerLoad concentrate, an optimized formulation of nonionic, Pluronic surfactant polyols, aids the solubilization of rhod-3 AM dye in physiological media and is available separately (P10200, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). Probenecid, also available separately (P36400, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), inhibits organic anion pumps that actively extrude the de-esterified dye.
With Ca2+ dissociation constants well above 1 µM, our relatively low-affinity Ca2+ indicators can be used to detect intracellular Ca2+ levels in the micromolar range—levels that would saturate the response of fluo-3 and rhod-2. Such elevated Ca2+ levels are generated by mobilization of intracellular Ca2+ stores and by excitatory stimulation of smooth muscle and neurons. Moreover, low-affinity indicators have faster ion dissociation rates, making them more suitable for tracking the kinetics of rapid Ca2+ fluxes than indicators with Kd values <1 µM.
Fluo-5F, Fluo-4FF, Fluo-5N and Mag-Fluo-4
Fluo-5F, fluo-4FF, fluo-5N and mag-fluo-4 are analogs of fluo-4 with much lower Ca2+-binding affinity, making them suitable for detecting intracellular Ca2+ levels in the 1 µM to 1 mM range. Fluo-5F, fluo-4FF, fluo-5N and mag-fluo-4 have Kdvalues for Ca2+ of ~2.3 µM, ~9.7 µM, ~90 µM and ~22 µM, respectively, as compared with fluo-4, which has a Kd for Ca2+ of ~345 nM (measured at 22°C using our Calcium Calibration Buffer Kits) (Figure 19.3.1). The temperature dependence of the Kdfor fluo-5F has been reported. These low Ca2+-binding affinities are ideal for detecting high concentrations of Ca2+ in the endoplasmic reticulum and neurons (), as well as for tracking Ca2+ flux kinetics. Fluo-5N has been found to be particularly useful for monitoring calcium oscillations in the sarcoplasmic reticulum. Like fluo-4, these indicators are essentially nonfluorescent in the absence of divalent cations and exhibit strong fluorescence enhancement with no spectral shift upon binding Ca2+ (Figure 19.3.11). Because mag-fluo-4 is less Ca2+/Mg2+ selective than fluo-5N, it is also useful as an indicator for intracellular Mg2+ levels (Fluorescent Mg2+ Indicators—Section 19.6). Fluo-5F (F14221, F14222), fluo-4FF (F23980, F23981), fluo-5N (F14203, F14204) and mag-fluo-4 (M14205, M14206) are available as cell-impermeant potassium salts or as cell-permeant AM esters. The AM esters are specially packaged as a set of 10 vials, each containing 50 µg.
Rhod-5N (Figure 19.3.8) has a lower binding affinity for Ca2+ than any other BAPTA-based indicator (Kd = ~320 µM, measured at 22°C using our Calcium Calibration Buffer Kits). These properties confer suitability for Ca2+ detection in environmental and food testing applications. Like the parent rhod-2 indicator, rhod-5N is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca2+. Furthermore, rhod-5N has very little detectable response to Mg2+ concentrations up to at least 100 mM. Rhod-5N is available as a cell-impermeant potassium salt (R14207).
Rhod-FF and X-Rhod-5F
The fluorinated analogs of rhod-2—rhod-FF (Figure 19.3.8) and X-rhod-5F —have intermediate Ca2+ sensitivity relative to rhod-2 and rhod-5N. Their Ca2+ dissociation constants (Kd) are 19 µM and 1.6 µM, respectively (measured at 22°C using our Calcium Calibration Buffer Kits). X-rhod-5F is available as a water-soluble potassium salt ( X23984), and rhod-FF and X-rhod-5F are available as cell-permeant AM esters (R23983, X23985). The AM esters are specially packaged as a set of 10 vials, each containing 50 µg.
Indicators of Mitochondrial Ca2+ Transients
Our AM esters of rhodamine-based indicators include:
These rhodamine derivatives form a set of cell-permeant Ca2+ indicators with a net positive charge (, ). This property promotes their sequestration into mitochondria in some cells, most likely via membrane potential–driven uptake, and results in a staining pattern that is characteristic of mitochondria (, , ). Mitochondria have a high capacity for Ca2+ uptake and therefore require low-affinity Ca2+ indicators to accurately measure internal Ca2+ concentrations. Because the range of Ca2+ concentrations that can be detected using rhod-2 AM is limited to within about one order-of-magnitude above and below its Ca2+ dissociation constant (Kd = 570 nM), we offer a selection of rhod-2 analogs with Ca2+ dissociation constants up to 320 µM (Summary of Molecular Probes fluorescent Ca2+ indicators—Table 19.1, Figure 19.3.8).
The extent of mitochondrial versus cytosolic localization is influenced by the temperature and incubation time used for AM ester loading. By reducing rhod-2 AM to the colorless, nonfluorescent dihydrorhod-2 AM, the discrimination between cytosolic and mitochondrially localized dye can be further improved. The AM ester of dihydrorhod-2 exhibits Ca2+-dependent fluorescence only after it is oxidized and its AM esters are cleaved to yield the rhod-2 indicator, processes that occur rapidly in the mitochondrial environment. A detailed protocol for reducing rhod-2 AM to generate dihydrorhod-2 AM is available (Preparation of Dihydrorhod-2 AM). The procedure should also be suitable for reduction of the AM esters of the other rhod indicators. Co-loading of cells with rhod-2 AM or X-rhod-1 AM in combination with green-fluorescent Ca2+ indicators such as fura-2, fluo-3, fluo-4, Calcium Green-1 and Oregon Green 488 BAPTA-1 enables simultaneous two-color imaging of mitochondrial and cytoplasmic Ca2+ (Figure 19.3.12). Mitochondrial Ca2+ and mitochondrial NADH can be simultaneously measured using rhod-2 and intrinsic NADH fluorescence, respectively.
Figure 19.3.12 Simultaneous measurement of intracellular and mitochondrial Ca2+ in pulmonary artery smooth muscle cells. Cells were loaded with 2 µM rhod-2 AM, R1244, R1245MP) for 60 minutes at 22°C and were subsequently patch-clamped for electrophysiological measurements. Fura-2 (50 µM; F1200, F6799) was loaded by infusion from the patch pipette; at the same time, residual cytosolic rhod-2 was dialyzed out. Fluorescence excitation was rapidly alternated between 340 nm and 380 nm (for fura-2) and 500 nm (for rhod-2). The Ca2+ transient was detected following application of 100 µM ATP for 10 seconds. The observed increases in mitochondrial Ca2+ may provide a feedback mechanism for matching ATP supply and demand by activating Ca2+-dependent dehydrogenases in the ATP synthesis pathway. This figure was reproduced with permission from J Physiol (1999) 516:139.
Calcium Green-1 and Calcium Green-2 Indicators
Invitrogen™ Calcium Green™-1 (Figure 19.3.13, ) and Calcium Green™-2 (Figure 19.3.14) indicators, as well as Calcium Orange™ and Calcium Crimson™ indicators (described below), are visible light–excitable indicators developed in our laboratories. Like fluo-3 and fluo-4, the Calcium Green indicators exhibit an increase in fluorescence emission intensity upon binding Ca2+ with little shift in wavelength (Figure 19.3.15, Figure 19.3.16); the fluorescence spectra of the Calcium Green indicators are almost identical to those of fluo-3. Further comparison of the Calcium Green indicators and fluo-3 reveals that, at high Ca2+ levels, Calcium Green-1 and Calcium Green-2 are several times brighter than fluo-3 (Figure 19.3.2). Calcium Green-1 has a quantum yield of 0.75 at saturating Ca2+ concentrations, as compared with about 0.14 for fluo-3.
The Calcium Green indicators have several other important features:
- Calcium Green-1 is more fluorescent in resting cells than is fluo-3 (Figure 19.3.2), which increases the visibility of unstimulated cells, facilitates the determination of baseline fluorescence and makes calculations of intracellular Ca2+ concentrations more reliable.
- Calcium Green-1 has been a preferred indicator for multiphoton excitation imaging of Ca2+ in living tissues.
- Calcium Green-1 is useful for measuring intracellular Ca2+ by fluorescence lifetime imaging (FLIM)
- The Ca2+ affinity of Calcium Green-1 in the absence of Mg2+ (Kd for Ca2+ = 190 nM) is higher than that of fluo-3 (Kd for Ca2+ = 390 nM) or Calcium Green-2 (Kd for Ca2+ = 550 nM, measured at 22°C using our Calcium Calibration Buffer Kits).
- Like fluo-3 and fluo-4, Calcium Green-2 is essentially nonfluorescent in the absence of Ca2+ and exhibits an approximately 100-fold increase in emission intensity upon Ca2+ binding, which leads to a very large dynamic range.
- Like fluo-3 AM and fluo-4 AM, the AM esters of the Calcium Green indicators are nonfluorescent.
Furthermore, the Calcium Green indicators are less phototoxic to cells than fluo-3. This observation stems at least in part from the fact that the Calcium Green indicators are intrinsically more fluorescent than fluo-3, thus requiring lower illumination intensities and lower dye concentrations to achieve the same signal (Figure 19.3.2).
Simultaneous loading of Calcium Green-2 and carboxy SNARF-1 AM, acetate (C1271, C1272; Probes Useful at Near-Neutral pH—Section 20.2) enabled researchers to make ratiometric measurements of intracellular Ca2+ in cardiac myocytes. A variation on this approach has been reported by Oheim and co-workers in which Calcium Green-1 and a Ca2+-insensitive reference dye similar to lucifer yellow CH (L453, L12926; Polar Tracers—Section 14.3) were loaded into bovine adrenal chromaffin cells by whole-cell patch clamping. Ratio images were obtained using 420/488 nm dual excitation (emission >515 nm) and exhibited superior signal-to-noise characteristics, as compared with conventional UV-excited fura-2 images. This technique should be equally applicable to other 488 nm–excited Ca2+ indicators such as fluo-3, fluo-4, fluo-5N and the Oregon Green 488 BAPTA series. Calcium Green-1 and Calcium Green-2 are available as cell-impermeant potassium salts (C3010MP, C3730) or as cell-permeant AM esters (C3011MP, C3012, C3732).
Figure 19.3.15 Ca2+-dependent fluorescence emission spectra of the Calcium Green-1 indicator (C3010MP).
Figure 19.3.16 Ca2+-dependent fluorescence emission spectra of the Calcium Green-2 indicator (C3730).
Calcium Orange and Calcium Crimson Indicators
Like the Calcium Green indicators, Calcium Orange (C3013, ) and Calcium Crimson exhibit an increase in fluorescence emission intensity upon binding to Ca2+ with little shift in wavelength (Figure 19.3.17, Figure 19.3.18) and can be loaded into cells as their AM esters (C3015, C3018). Both the Calcium Orange and Calcium Crimson indicators are more photostable than either fluo-3 or the Calcium Green indicators.
The excitation maximum (~590 nm) of Calcium Crimson makes it useful in situations where interference by cellular autofluorescence is problematic. Selective uptake of Calcium Crimson AM (C3018) at Drosophila motor nerve terminals has been used to obtain images of stimulus-dependent Ca2+ influx.
Figure 19.3.17 Ca2+-dependent fluorescence emission spectra of the Calcium Orange indicator (C3013).
Figure 19.3.18 Ca2+-dependent fluorescence emission spectra of the Calcium Crimson indicator (available as the cell-permeant acetoxymethyl (AM) ester, C3018).
Calcium Green-5N and Magnesium Green: Low-Affinity Ca2+ Indicators
The Ca2+ indicators Invitrogen™ Calcium Green™-5N (Figure 19.3.13) and Magnesium Green™ () have dissociation constants for Ca2+ in the absence of Mg2+ of ~14 µM and ~6 µM, respectively (measured at 22°C using our Calcium Calibration Buffer Kits) (Figure 19.3.19). These low-affinity Ca2+ indicators exhibit relatively little fluorescence, except in cells in which high-amplitude Ca2+ influx or release is occurring. Calcium Green-5N and Magnesium Green buffer intracellular Ca2+ to a lesser extent than do the higher-affinity Ca2+ indicators. Furthermore, the high Ca2+ dissociation rates of these indicators are advantageous for tracking rapid Ca2+-release kinetics. Use of the low-affinity Ca2+ indicator Calcium Green-5N in combination with the higher-affinity indicator Calcium Green-2 in the same experimental protocol can give an indication of the absolute magnitude of Ca2+ spikes. Furthermore, coinjection of Ca2+-sensitive Calcium Green-5N and Ca2+-insensitive 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS, A350; Polar Tracers—Section 14.3) into Limulus ventral nerve photoreceptors permitted ratiometric measurement of Ca2+ flux.
Calcium Green-5N is available as a cell-impermeant potassium salt (C3737) or as a cell-permeant AM ester (C3739). Magnesium Green, which is also discussed in Fluorescent Mg2+ Indicators—Section 19.6 with the other Mg2+indicators, is available as a cell-impermeant potassium salt (M3733) or as a cell-permeant AM ester (M3735).
Figure 19.3.19 Ca2+-dependent fluorescence emission spectra of the Calcium Green-5N indicator (C3737).
Invitrogen™ Oregon Green™ 488 BAPTA indicators are based on our exceptionally bright Oregon Green™ 488 dyes (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5). The absorptivity of Oregon Green 488 BAPTA-1 (Figure 19.3.13) at 488 nm is ~93% of its peak value, whereas the absorptivity of fluo-3 and the Calcium Green indicators at 488 nm is only ~45% of their maxima. Consequently, the Oregon Green 488 BAPTA indicators are more efficiently excited by the 488 nm spectral line of the argon-ion laser than are the fluo-3 and Calcium Green indicators.
Oregon Green 488 BAPTA-1 and Oregon Green 488 BAPTA-2
The spectral properties of the Oregon Green 488 BAPTA indicators permit the use of lower dye concentrations when using argon-ion laser excitation sources, making the Oregon Green 488 BAPTA indicators well suited for intracellular Ca2+ measurements by confocal laser-scanning microscopy. Oregon Green 488 BAPTA-1 is also recommended for fluorescence lifetime imaging (FLIM) and applications involving photoactivatable ("caged") probes ().
Furthermore, the quantum yields of the Ca2+ complexes of Oregon Green 488 BAPTA-1 and Calcium Green-1 are ~0.7, as compared with only ~0.14 for fluo-3. As with Calcium Green-1 (Figure 19.3.2), Oregon Green 488 BAPTA-1 is moderately fluorescent in Ca2+-free solution, and its fluorescence is enhanced about 14-fold at saturating Ca2+ (Figure 19.3.20). In some cases, it may be advantageous to mix Oregon Green 488 BAPTA-1 and fluo-4 indicators to obtain a calcium response that combines a finite basal signal level with a large stimulus-dependent increase. Oregon Green 488 BAPTA-1 has a Kd for Ca2+ in the absence of Mg2+ of about 170 nM. Oregon Green 488 BAPTA-2 is similar to Calcium Green-2 in that it contains two dye molecules per BAPTA chelator (Figure 19.3.14) and exhibits very low fluorescence in the absence of Ca2+. The fluorescence of Oregon Green 488 BAPTA-2 is enhanced at least 37-fold at saturating Ca2+, and it has a Kd for Ca2+ in the absence of Mg2+ of ~580 nM (Figure 19.3.21) (Kd values for Ca2+ are determined at ~22°C using our Calcium Calibration Buffer Kits).
Several research groups are exploiting the power of two-photon laser-scanning microscopy and fluorescent Ca2+ indicators for functional imaging of neurons in brain slice preparations and intact live brains. Oregon Green 488 BAPTA-1 and Calcium Green-1 (see above), excited in the wavelength range 800–850 nm are the indicators of choice in many of these investigations. The Oregon Green 488 BAPTA-2 Ca2+ indicator and red-fluorescent, Ca2+-insensitive sulforhodamine 101 reference dye (S359, Polar Tracers—Section 14.3) have been loaded together into optic nerves to image Ca2+ distribution in axons.
Oregon Green 488 BAPTA-1 and Oregon Green 488 BAPTA-2 are available as cell-impermeant potassium salts (O6806, O6808) or as cell-permeant AM esters (O6807, O6809), which are specially packaged as a set of 10 vials, each containing 50 µg. A 10,000 MW dextran conjugate of the Oregon Green BAPTA-1 indicator (O6798) is described in Fluorescent Ca2+ Indicator Conjugates—Section 19.4.
Figure 19.3.20 Ca2+-dependent fluorescence emission spectra of the Oregon Green 488 BAPTA-1 indicator (O6806).
Figure 19.3.21 Ca2+-dependent fluorescence emission spectra of the Oregon Green 488 BAPTA-2 indicator (O6808).
Oregon Green 488 BAPTA-6F and Oregon Green 488 BAPTA-5N
Oregon Green 488 BAPTA-6F (Figure 19.3.13) and Oregon Green 488 BAPTA-5N (Figure 19.3.13) are low-affinity Ca2+ indicators (Kd for Ca2+ ~3 µM and ~20 µM, respectively, measured at ~22°C using our Calcium Calibration Buffer Kits) designed for measuring intracellular Ca2+ levels above 1 µM. By simultaneously imaging Oregon Green 488 BAPTA-5N and rhod-2, researchers have been able to compare cytosolic and mitochondrial Ca2+responses to action potentials in amphibian motor nerve terminals. Oregon Green 488 BAPTA-6F and Oregon Green BAPTA-5N are available as cell-impermeant potassium salts (O23990, O6812).
Invitrogen™ Fura Red™ indicator (), a visible light–excitable fura-2 analog, offers unique possibilities for ratiometric measurement of Ca2+ in single cells by microphotometry, imaging or flow cytometry. The visible-wavelength excitation (450–500 nm) and very long-wavelength emission maximum (~660 nm) of the Fura Red indicator () minimize interference from autofluorescence and pigmentation in tissues and biological fluids. Fluorescence of the Fura Red indicator excited at 488 nm decreases once the indicator binds Ca2+ (). Even in the absence of Ca2+, fluorescence of the Fura Red indicator is much weaker than that of the other visible light–excitable Ca2+ indicators, necessitating use of higher concentrations of the indicator in cells to produce equivalent fluorescence. Fura Red is available as either a cell-impermeant tetrapotassium salt (F14219) or as a cell-permeant AM ester (F3020, F3021).
Ratiometric measurements of intracellular Ca2+ levels with Fura Red have been made using excitation wavelengths of 420 nm and 480 nm or 457 nm and 488 nm. A simultaneous assay for Ca2+ uptake and ATP hydrolysis by sarcoplasmic reticulum has been developed that uses the large absorbance change of Fura Red upon Ca2+ binding. This assay can measure Ca2+ uptake—and probably uptake of heavy metal ions through channels—from the medium in real time without the use of radioactive Ca2+, making it generally useful for measuring Ca2+ uptake by cells. In several cell types, simultaneous labeling with Fura Red and fluo-3 (Figure 19.3.4) has enabled researchers to use ratiometric measurements for estimating intracellular Ca2+ levels using confocal laser-scanning microscopy () or flow cytometry.
Furthermore, the huge Stokes shift of Fura Red permits multicolor analysis of Fura Red fluorescence in combination with fluorescein or fluorescein-like dyes using only a single excitation wavelength (Figure 19.3.4). For example, researchers have been able to simultaneously measure Ca2+ fluxes and oxidative bursts in monocytes and granulocytes by simultaneously measuring the fluorescence of Fura Red and rhodamine 123—the oxidation product of the probe dihydrorhodamine 123 (D632, D23806; Generating and Detecting Reactive Oxygen Species—Section 18.2). Fura Red has also been used in combination with blue-fluorescent protein in transfected cells.
Calcein (C481, Fluorescent Indicators for Zn2+ and Other Metal Ions—Section 19.7) is a relatively low-affinity Ca2+ chelator. The dissociation constants for both the Ca2+ and Mg2+ complexes of calcein at physiological pH are about 10-3 to 10-4 M. As a derivative of iminodiacetic acid (), this dye exhibits an ion affinity that increases considerably at higher pH, and thus it is not particularly useful for measuring Ca2+ or Mg2+ in cells. Instead, it is primarily useful for detecting mineralized Ca2+, particularly in the context of bone and bacterial spores. Calcein is also widely used for fluorescence quenching–based detection of Ni2+, Co2+, Cu2+ and Fe3+ (Fluorescent Indicators for Zn2+ and Other Metal Ions—Section 19.7).
|Low Ca2+||High Ca2+|
|C3010MP||1147.19||F,D,L||pH >6||506||81,000||531||H2O||506||82,000||531||H2O/Ca2+||190 nM||1, 2, 3, 4|
|C3013||1087.33||F,D,L||pH >6||549||80,000||575||H2O||549||80,000||576||H2O/Ca2+||185 nM||1, 2, 3, 4|
|Calcium Crimson||1232.51||F,D,L||pH >6||589||96,000||615||H2O||589||92,000||615||H2O/Ca2+||185 nM||1, 2, 3, 4|
|C3730||1665.58||F,D,L||pH >6||506||95,000||536||H2O||503||147,000||536||H2O/Ca2+||550 nM||1, 2, 3, 4|
|C3737||1192.19||F,D,L||pH >6||506||83,000||532||H2O||506||82,000||532||H2O/Ca2+||14 µM||1, 2, 4, 5|
|F1240||854.70||F,D,L||pH >6||503||92,000||see Notes||H2O||505||102,000||526||H2O/Ca2+||390 nM||1, 2, 3, 6|
|F3715||960.00||F,D,L||pH >6||506||90,000||see Notes||H2O||506||100,000||526||H2O/Ca2+||390 nM||1, 2, 3, 6|
|F14200||927.09||F,D,L||pH >6||491||82,000||see Notes||H2O||494||88,000||516||H2O/Ca2+||345 nM||1, 2, 3, 6|
|F14203||958.06||F,D,L||pH >6||491||72,000||see Notes||H2O||493||74,000||518||H2O/Ca2+||90 µM||1, 2, 5, 6|
|F14217||1096.95||F,D,L||DMSO||456||26,000||see Notes||MeOH||F14200||7, 8|
|F14218||1129.86||F,D,L||DMSO||464||26,000||see Notes||MeOH||F1240||7, 8|
|F14219||808.98||F,D,L||pH >6, MeOH||473||29,000||670||H2O||436||41,000||655||H2O/Ca2+||140 nM||1, 2, 3, 9|
|F14221||931.05||F,D,L||pH >6||491||71,000||see Notes||H2O||494||74,000||518||H2O/Ca2+||2.3 µM||1, 2, 5, 6|
|F23915||1129.86||F,D,L||DMSO||464||26,000||see Notes||MeOH||F1240||7, 10|
|F23917||1096.95||F,D,L||DMSO||456||26,000||see Notes||MeOH||F14200||7, 10|
|F23980||949.04||F,D,L||pH >6||491||72,000||see Notes||H2O||494||75,000||516||H2O/Ca2+||9.7 µM||1, 2, 5, 6|
|F36201||1055.26||F,D,L||pH >6||491||74,000||see Notes||H2O||494||78,000||518||H2O/Ca2+||950 nM||1, 2, 3, 6|
|M3733||915.90||F,D,L||pH >6||506||77,000||531||H2O||506||77,000||531||H2O/Ca2+||6 µM||1, 2, 4, 5|
|M14205||681.77||F,D,L||pH >6||490||74,000||see Notes||H2O||493||75,000||517||H2O/Ca2+||22 µM||1, 2, 5, 6|
|O6806||1114.28||F,D,L||pH >6||494||76,000||523||H2O||494||78,000||523||H2O/Ca2+||170 nM||1, 2, 3, 4|
|O6808||1599.77||F,D,L||pH >6||494||105,000||523||H2O||494||140,000||523||H2O/Ca2+||580 nM||1, 2, 3, 4|
|O6812||1159.28||F,D,L||pH >6||494||72,000||521||H2O||494||76,000||521||H2O/Ca2+||20 µM||1, 2, 4, 5|
|O23990||1132.27||F,D,L||pH >6||494||75,000||523||H2O||494||77,000||523||H2O/Ca2+||3 µM||1, 2, 4, 5|
|R14207||900.03||F,D,L||pH >6||549||64,000||see Notes||H2O||551||63,000||577||H2O/Ca2+||320 µM||1, 2, 6, 12|
|R14220||869.06||F,D,L||pH >6||548||91,000||see Notes||552||96,000||578||H2O/Ca2+||570 nM||1, 2, 3, 6|
|rhod-FF||891.02||F,D,L||pH >6||548||73,000||see Notes||H2O||552||78,000||577||H2O/Ca2+||19 µM||1, 2, 5, 6|
|X-rhod-1||973.21||F,D,L||pH >6||576||88,000||see Notes||H2O||580||92,000||602||H2O/Ca2+||700 nM||1, 2, 3, 6|
|X23984||977.18||F,D,L||pH >6||576||73,000||see Notes||H2O||580||73,000||602||H2O/Ca2+||1.6 µM||1, 2, 5, 6|