Advantages of calcium green-1 over other fluorescent dyes in measuring cytosolic calcium in platelets.

Fluorescent indicators are widely used in the measurements of cytosolic calcium in many cell types for many purposes because they are relatively easy to use. Notwithstanding, they have some defects to prevent accurate measurements under certain conditions, such as significant dye leakage and UV-quenching effect. Menadione, a representative quinone derivative with antiaggregating effect, is also UV-absorbent. To investigate whether menadione can affect the change of cytosolic calcium in platelets by agonist, we measured the change of cytosolic calcium level using calcium green-1. Since this dye has not been used previously in platelets, we determined that the optimal loading of calcium green-1 to platelets was achieved using 3 microM dye incubated for 60 min at 37 degrees C. Our study compared the use of calcium green-1 with fura-2 and fluo-3 (two widely used dyes) in measurements of cytosolic calcium. Fura-2 is UV-excited, so when menadione was treated in fura-2-loaded cells, it had a quenching effect. Fluo-3, the other visible fluorescent indicator, leaked from platelets very rapidly and required the use of anion channel blockers which are known to affect physiological response of platelets. Our study demonstrated that changes in cytosolic calcium levels can be accurately measured without these problems by using calcium green-1. We therefore were able to demonstrate that menadione inhibited calcium increase by thrombin in a dose-dependent manner similar to menadione's antiaggregating effect in platelets.     

Simultaneous measurement of intracellular Ca(2+) and nitric oxide: a new method

Different methods to measure the unstable radical nitric oxide (NO) have been established. We are going to present a new method to measure intracellular calcium and NO simultaneously in endothelial cells. A new fluorescent dye (DAF-2) has been developed recently which binds NO resulting in an enhanced fluorescence. We loaded porcine aortic endothelial cells with Fura-2, a fluorescent dye commonly used to measure intracellular calcium, and DAF-2 simultaneously (cell permeable dyes). Using excitation wavelengths of lambda 340 nm (Fura-2) and lambda 485 nm (DAF-2) we could show that thrombin induces an intracellular calcium increase and simultaneously a NO formation in endothelial cells which could be blocked by a NO synthase inhibitor. This new method of a simultaneous measurement of intracellular calcium and NO provides the possibility to follow intracellular calcium and NO distributions online, and is sensitive enough to monitor changes of NO formed by the constitutive endothelial NO-synthase.     

Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers

Fura-2 is widely used to measure the concentration of cytosolic free calcium, but in many cells the dye does not remain localized within the cytoplasmic matrix. In these cells, Fura-2 is sequestered within intracellular organelles, secreted into the extracellular medium, or both. We have found that, in mouse peritoneal macrophages, J774 cells, PC12 cells, and N2A cells, Fura-2 sequestration and secretion are mediated by organic anion transport systems and are blocked by the inhibitors probenecid and sulfinpyrazone. Under appropriate conditions these agents have little affect on calcium transients, and may facilitate the use of Fura-2 in a variety of cell types.     

Intracellular Ca2+ measurement with Indo-1 in substrate-attached cells: advantages and special considerations

The dual emission, Ca2+ sensitive fluorescent dye, Indo-1, offers several potential advantages over its dual excitation analogue, Fura-2. Most notable among these advantages are increased speed of measurement using dual wavelength photometry and the absence of a requirement for special quartz optics. Despite these potential advantages, only a tiny fraction of the microscopic studies of intracellular free calcium ([Ca2+]i) on substrate-attached cells has employed Indo-1. Among the reasons for the infrequent use of Indo-1 are the fact that it exhibits somewhat different spectral properties in the cytosol than it does in extracellular buffers, and the notion that it is much more sensitive to photobleaching than Fura-2. We report here that under our experimental conditions, Indo-1 photobleaching is small and does not noticeably affect the measurement of free Ca2+, even after 30 minutes of continuous illumination. We also report a new method for creating in situ standard curves that is easy, reproducible, and yields values for [Ca2+]i that are identical to those obtained with Fura-2. In addition, we have found that Indo-1 is less subject than Fura-2 to compartmentalization within subcellular organelles. These results provide baseline data to take advantage of the significant improvement afforded by Indo-1 in the measurement of rapid [Ca2+]i responses and the avoidance of compartmentalization artifacts during experiments of long duration.     

Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions

Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types.     

Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) partially inhibits rapid intracellular free calcium transients triggered by anti-immunoglobulin in murine lymphocytes

The highly fluorescent probes Indo-1 and Fura-2 were employed to detect intracellular calcium responses in murine splenic lymphocytes following cross-linking of cell surface Ig. Inhibition by phorbol ester (12-O-tetradecanoylphorbol 13-acetate) was rapid and showed a strong preference for the very transient phase of the response which has been identified as a mobilization of intracellular calcium. 12-O-Tetradecanoylphorbol-13-acetate had significantly less effect on the longer lasting increase in intracellular free calcium which involved an influx of extracellular calcium. Whole spectra were used as a check on transients, which were monitored at a single wavelength, in order to eliminate changes that were not calcium-dependent. It was found that such changes could arise from the association of Indo-1, or its acetoxymethyl ester, with phospholipid bilayers since this affected their fluorescence spectra. In addition, the loading of resting cells with dye esters was shown to be greatly enhanced by the inclusion of a small amount of the detergent Pluronic F-127 in the incubation medium. A spectral analysis of labeled cells showed that the extent of hydrolysis of intracellular dye was improved as well as the rate of uptake by cells.     

The estimation of free calcium within synaptosomes and mitochondria with fura-2; comparison to quin-2

The utility of the acetoxymethyl esters of two tetracarboxylic acids, fura-2 and quin-2, in the determination of ionic calcium levels within synaptosomes and mitochondria was compared. Synaptosomes and isolated mitochondria both accumulated the esters but mitochondria had a much more limited capacity to hydrolyze them. Dye-loaded synaptosomes maintain their external membrane potential of magnitude similar to values for unloaded controls and do not accumulate radioactive Ca²⁺ in excess with time. Both fluorescent compounds yielded similar values (about 300–400 nM) for free intrasynaptosomal calcium [Ca²⁺]_i. Mitochondrial Ca²⁺ could be measured only with fura-2. Isolated mitochondria contained 0.9–1 μM free Ca²⁺ in a similar extrasynaptosomal medium. Fura-2 tended to overestimate [Ca²⁺]_i while quin-2 tended to underestimate it due to chelation of these dyes with intrasynaptosomal trace elements. Fura-2 requiring the use of two excitation wavelengths was significantly superior to the single wavelength method using quin-2. Advantages included reduced danger of erroneous readings due to (i) synaptosomal sedimentation, (ii) photobleaching of the dye, (iii) underestimation of intrasynaptosomal calcium during correction for dye leakage by manganese entry into synaptosomes. Fura-2 interfered less with synaptosomal Ca²⁺ transients than quin-2, probably due to lower intrasynaptosomal concentration of dye needed. Both unstimulated and K⁺-stimulated ⁴⁵Ca²⁺ uptake were increased in quin-2-loaded synaptosomes but only K⁺-stimulated uptake in fura-2 loaded ones. This series of advantages makes fura-2 of superior utility in the determination of free intrasynaptosomal calcium.     

Intracellular calcium during chemotaxis of Dictyostelium discoideum: a new fura-2 derivative avoids sequestration of the indicator and allows long-term calcium measurements.

During stimulation of Dictyostelium discoideum amoebae with the chemoattractant cAMP, extracellular calcium is taken up by the cells. The aim of this study was to determine the cytosolic free calcium concentration ([Ca++]i) during chemotaxis of Dictyostelium cells. In contrast to most vertebrate cells, three major drawbacks were encountered: 1) the indicator fura-2 could not be introduced into the cells by incubation with the ester form, 2) once loaded, the dye was rapidly sequestered into vesicles, 3) the organic anion transport blocker probenecid was not suitable to block sequestration. These problems were met by introducing the indicator into the cells with the scrape-loading technique adapted for use with Dictyostelium and the construction of a new fura-2 derivative, fura-2-dextran. Scrape-loading of Dictyostelium yielded up to 40% of labeled, vital cells. Fura-2-dextran fulfilled the following criteria: 1) it remained homogeneously distributed in the cytoplasm of motile Dictyostelium cells, 2) it retained the fluorescence intensity of fura-2 and the affinity for calcium binding, 3) it was very well suitable to demonstrate changes of [Ca++]i in serum-stimulated fibroblasts. [Ca++]i-measurements with fura-2-dextran in chemotactically active D. discoideum amoebae revealed that the large decrease in the extracellular calcium concentration is not accompanied by an overall change in [Ca++]i. Chemotaxis in this organism occurs in the absence of global changes in [Ca++]i. However, we cannot exclude either short-lived or local changes just beneath the plasma membrane.     

The separate roles of calcium and mechanical forces in mediating cell death in mechanically injured neurons

In this study, we examine the response of cultured hippocampal neurons to mechanical stretch. To measure the immediate response, we measured the response of a calcium sensitive fluorescent dye (Fura-2AM) prior to and for five minutes following stretch. In separate experiments, we measured the viability of the cells using propidium iodide labeling at 24 hours following a single stretch. Results show that cytosolic calcium increases immediately following stretch to levels much higher than those associated with maximal chemical agonist (NMDA) stimulation. Moreover, the calcium response data indicate a loss in calcium regulation in the neurons at the most severe level of stretch. Finally, at the highest levels of stretch, the cell survivability decreases at 24 hrs following the stretch injury. These data show the association between loss in calcium regulation and early signs of neuronal cell death.     

Transient increases of intracellular Ca2+ induced by volatile anesthetics in rat hepatocytes

The affects of volatile anesthetics on mobilization of intracellular Ca2+ was monitored in primary cultures of rat hepatocytes using the fluorescent Ca2+ probe Fura-2. The use of Fura-2 was limited by several factors which complicated the quantitative analysis of the results, such as: (i) a high rate of dye leakage; (ii) changes in the redox state of the hepatocytes which interfered with the fluorescence produced by the dye at various excitation wavelengths; (iii) compartmentalization of the dye producing high local intracellular concentrations; and, of particular importance for this study, (iv) enhanced photobleaching of the dye in the presence of halothane. To aid in the interpretation of the Fura-2 data, the Ca2(+)-sensitive photoprotein aequorin was also used to monitor changes in [Ca2+]i. The aequorin and Fura-2 techniques qualitatively yielded the same result, that the volatile anesthetic agents halothane, enflurane, and isoflurane induce an immediate and transient increase of [Ca2+]i. The durations of these transients were approximately between 5 and 10 min and were not related to any evident acute cell toxicity. The [Ca2+]i increases induced by the volatile anesthetic agents were dose-dependent, with halothane the most potent. The exact mechanism governing these increases in [Ca2+]i induced by these anesthetics in rat hepatocytes is unknown, but is likely to involve effects on both the cell surface membrane and endoplasmic reticulum components of the signal transducing system.     

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.     

Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

Plasma membrane Ca²⁺-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca²⁺]_i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca²⁺]_i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.     

Fluorescence combined with excised patch: measuring calcium currents in plant cation channels

Combined application of the patch–clamp technique and fura-2 fluorescence detection enables the study of study calcium fluxes or related increases in cytosolic calcium concentration. Here we used the excised patch configuration, focusing the photomultiplier on the tip of the recording pipette where the fluorescent dye was present (FLEP, fluorescence combined with excised patch). This configuration has several advantages, i.e. a lack of delay in loading the fluorophore, of interference by internal calcium buffers and of photobleaching, due to the quasi-infinite dye reservoir inside the pipette. Upon voltage stimulation of tonoplast patches, sustained and robust fluorescence signals indicated permeation of calcium through the slow vacuolar (SV) channel. Both SV currents and fluorescence signal changes were absent in the presence of SV channel inhibitors and in vacuoles from Arabidopsis tpc1 knockout plants that lack SV channel activity. The fractional calcium currents of this non-selective cation channel were voltage-dependent, and were approximately 10% of the total SV currents at elevated positive potentials. Interestingly, calcium permeation could be recorded as the same time as oppositely directed potassium fluxes. These events would have been impossible to detect using patch–clamp measurements alone. Thus, we propose use of the FLEP technique for the study of divalent ion-selective channels or transporters that may be difficult to access using conventional electrophysiological approaches.     

Free calcium concentrations in bullfrog rods determined in the presence of multiple forms of Fura-2.

We employed the fluorescent calcium indicator Fura-2, loaded into intact retinas of the bullfrog Rana catesbeiana, to measure free calcium concentrations in the rod outer-segment cytosol. We determined that traditional methods of calculation yielded erroneous values of calcium. This error results from the presence of at least two distinct pools of Fura-2 in rod outer segments. Application of manganese quenches each pool, but quenching occurs at different rates. Using this fact, we show that the pools can be isolated by brief exposure to manganese and examined separately. One of these pools has the same fluorescent properties as the free salt of Fura-2 we use in our in vitro calibrations. The other source of fluorescence has more unusual properties. Although insensitive to calcium concentrations in the physiological range, it contributes significant anomalous fluorescence when cytosolic free calcium concentrations are elevated by application of IBMX. Nevertheless, the experimentally isolated, classic pool of Fura-2 is well behaved and allows us to calculate calcium concentrations relative to the Kd of Fura-2 by the usual ratio method. We show that when rods are exposed to saturating light, the free calcium concentration in their outer segments falls to a level not significantly different from zero within 20-30 s.     

Measurement of ionized calcium in normal human blood platelets

To measure the concentrations of cytosolic ionized calcium in platelets we used a calcium-sensitive fluorophor (Fura-2) with two different spectrofluorometers (Perkin-Elmer LS-5 and Fluorolog 222). Values obtained by these two instruments for the basal cytosolic ionized calcium concentration of resting platelets and those of agonist-activated platelets did not differ significantly. Both instruments were capable of monitoring the shifts in wavelengths induced by the dye-calcium complex, the ratio between absorbances at the two wavelengths (340/380 nm), and calcium concentrations continuously during agonist-induced platelet activation. We conclude that a relatively inexpensive instrument may be adequate for measuring ionized calcium in cells by this method, although sophisticated kinetic studies may require analytical or research-grade instruments.     

Distribution of intracellular free calcium in quiescent BALB/c 3T3 cells stimulated by platelet-derived growth factor

A digital imaging microscope and fluorescent Ca(2+)-sensitive probe (Fura 2) were used to study the spatial location and time course of increases in free intracellular calcium (Cai) induced by platelet-derived growth factor (PDGF). Microinjection of Fura 2 acid avoided problems of incomplete deesterification of Fura 2-acetoxymethyl ester (Fura 2/AM) and dye localization in cellular organelles. PDGF stimulated a rapid increase in Cai (up to 8-fold increase) in both the nucleus and the cytoplasm in approximately half of the quiescent BALB/c 3T3 cells. Cai changes were both spatially and temporally heterogeneous, the latter including both transient (1-2 min) and prolonged increases (greater than 5 min) in the same cell. PDGF stimulated mitogenesis and Cai increases in approximately the same percentage of cells. Moreover, large intracellular concentrations of a Ca2+ buffer (Quin 2) inhibited both Cai increases and mitogenesis stimulated by PDGF. Thus, Ca2+ increases in the nuclear and/or cytosolic compartments appear to be required for the stimulation of mitogenesis by polypeptide growth factors such as PDGF.     

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.     

Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.     

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.Download video file.^{(73M, flv)}     

Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2

The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.