首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Spectral evidence for non-calcium interactions of intracellular Indo-1   总被引:3,自引:0,他引:3  
Indo-1 is widely used to measure intracellular free calcium, [Ca2+]i, by comparing the fluorescence emission at 2 or more wavelengths with the emissions, which are assumed to be known, of Indo-1 when it is fully calcium-bound and when it is fully calcium-free. Accurate quantitation requires that these "reference" values be obtained on intracellular dye, and the full spectra of this study show that the reason is a significant spectral shift of the calcium-free peak, but not the calcium-bound. A mathematical analysis shows that the new peak must be a new state of the Indo-1 molecule, since it cannot be simply due to residual calcium in the cell. When intracellular "reference" spectra were used in the data analysis, [Ca2+]i could be calculated from whole spectra or from the ratio of observations at two wavelengths with good agreement. When extracellular "reference" spectra were used, the value calculated by the ratio method depended on the choice of wavelengths.  相似文献   

2.
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.  相似文献   

3.
Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.  相似文献   

4.
The dyes carboxy-SNARF-1 and BCECF are fluorescent probes of intracellular pH that exhibit changes in spectral shape upon proton binding which allow one to use measurements of fluorescence at two or more wavelengths in order to measure pH without artifacts associated with variability in dye loading, etc. In evaluating these dyes for this study, whole spectra, rather than measurements at two wavelengths, were analyzed. For BCECF, the effects of the intracellular milieu were minimal: both the pH-sensitive excitation spectrum and the pKa agreed closely with values found in extracellular solution. In contrast, both the spectra and the pKa for the emission spectrum-shifting carboxy-SNARF-1 showed significant differences between intracellular and extracellular dye. As a result, extremely misleading values for intracellular pH will be obtained if one attempts to use extracellular dye to calibrate intracellular carboxy-SNARF-1 measurements. Multiple origins were found for the discrepancy: (i) the intracellular dye was found to be significantly quenched, with the deprotonated form being more strongly quenched than the protonated form; and (ii) the pKa for the equilibrium with intracellular hydrogen ions was shifted by +0.2 pH units. These effects were readily reversed by disruption of the cell, but were not due to sequestering of dye in an acidic cell compartment.  相似文献   

5.
Assessment of Fura-2 for measurements of cytosolic free calcium   总被引:21,自引:0,他引:21  
Fura-2 has become the most popular fluorescent probe with which to monitor dynamic 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) accurate 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 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies 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 calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, 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.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

6.
It is assumed that the spectra of fluorescent probes indo-1 and fura-2 in the cytoplasm are linear combinations of the spectra of calcium-bound and free probes with weight factors proportional to the concentrations of these forms. When the concentration of calcium is measured by the dual-wavelength method, the above assumption is employed without testing. A multiwavelength method for measuring free cytosolic calcium concentration is described in the present study. The method is based on the registration of the fluorescence spectra of the probe with an optical multichannel analyzer and deconvolution of the spectra into components, corresponding to free and bound forms of the probe. A mismatch is also calculated to allow estimation of deconvolution accuracy. It was found that the spectra, recorded in aqueous calibration solution with varying calcium concentrations, can be deconvoluted into components, obtained both in the absence of calcium and at its saturating concentration. When the spectrum of the probe in the cytoplasm is deconvoluted into the same components the mismatch is higher. When aqueous calibration is used, the cytosolic calcium concentration determined by the dual-wavelength method is dependent considerably on the selected wavelengths. Our data indicate that this phenomenon may be associated with the lower polarity of cytoplasm compared to the aqueous calibration solution. Addition of either ethanol or glycerol into the calibration medium results in a considerable decrease in the mismatch. The optimal concentration of ethanol is 22-32%, and depends on the type and condition of cells tested. It is shown that the use of calibration spectra obtained in aqueous solutions leads to considerable overestimation of cytosolic calcium concentration.  相似文献   

7.
In terms of accuracy and sensitivity, intracellularly trapped, pH-dependent fluorescent probes are appropriate to accurately measure intracellular pH. These probes are commonly introduced into living cells in esterified form, wherein the free acid is produced through enzymatic hydrolysis. The fluorescence characteristics of the ester and the free acid can differ markedly and spectral uncertainty can occur. We describe here the measurement of intracellular pH using 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) that has been scrape-loaded into BALB/c-3T3 mouse cells. The excitation spectrum of pyranine is pH sensitive, with an isosbestic point at 415 nm and peaks at 405 and 465 nm which decrease and increase with pH, respectively. The 465/405 ratio can be used to monitor the pH, while the fluorescence at 415 nm indicates the total dye-dependent signal remaining. The scrape-loaded dye persists in cells for periods up to 6 h. We have calibrated this dye in situ using nigericin/high K+, and have found that the pKa of the dye in situ is 7.82, as compared to 7.68 in vitro. We have observed that the cells can slowly equilibrate their intracellular pH to near control levels when presented with either an acute alkaline or acid load.  相似文献   

8.
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.  相似文献   

9.
Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2's (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.  相似文献   

10.
A multiwavelength method for measuring free cytosolic calcium concentration is proposed. It is based on the registration of the fluorescent spectrum of calcium--sensitive probe indo-1 and deconvolution of the spectrum into components corresponding to free and bound forms of the probe. Calcium concentration is calculated as a product of calcium-probe dissociation constant by calcium-bound to free form concentration ratio. The obtained values are independent of variations in light-scattering properties of the medium and total dye concentration in the optical channel. It is shown that during ADP-induced platelet aggregation calcium concentration rises without measurable delay after the addition of the inducer and significantly decreases by the time the aggregation begins.  相似文献   

11.
Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca(2+)-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca(2+)-free and Ca(2+)-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K(eff)) and dynamic ranges (R(f)) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K(eff) = 181 +/- 52 nM, 1.16 +/- 0.016 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; and R(f) = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.  相似文献   

12.
V Kachel  O Kempski  J Peters  F Sch?del 《Cytometry》1990,11(8):913-915
Recently, new fluorescent dyes have been introduced into flow cytometry which alter their spectral characteristics when changes occur in certain cell features, e.g., intracellular pH or calcium ion concentration. Such changes may be determined by measuring the fluorescence intensity ratio in two different wavelength ranges (5). Here a new method is described, which simplifies the use of steadily flowing fluids for calibration. The pulse electronics of a flow cytometer cannot process the static fluorescence signals of a streaming fluid. If, however, the exciting or emitted fluorescence light of a calibration fluid is made pulsating, the flow cytometer electronics can evaluate those pulses. The new calibration procedure uses measurement of two wavelength windows shown in a two-parameter display to generate an absolute calibration scale. Measurement of the spectral shift in calibration fluids under identical instrumental settings provides absolute values that measurements of intracellular concentrations can be referred to.  相似文献   

13.
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.  相似文献   

14.
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)  相似文献   

15.
R Y Tsien  T J Rink  M Poenie 《Cell calcium》1985,6(1-2):145-157
Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.  相似文献   

16.
J Chambron  R Bidet  G Weill 《Biopolymers》1971,10(2):225-242
The desorption and melting with temperature of proflavine–DNA complexes has been studied by spectrophotometry and spectrofluorometry. Two methods are described to determine at each temperature the concentration of free and bound dye. The first one is based on the quenching of fluorescence of the free dye by the iodine ion, the second on fluorescence polarization measurements. It is shown that the sites where the bound dye fluoresces are thermally less stable than those where it is quenched, in such a way that a redistribution of the dye between the two types of sites occurs at intermediate temperatures, leading to a drop in the total fluorescence. This confirms the nature of the “emitting” sites which correspond to AT-rich region, while “quenched” sites correspond to GC-rich region. The first have a larger binding constant at room temperature, but only the latter are stabilized by dye intercalation. The desorption and melting have also been followed through the relative changes of absorption. The curves obtained at different wavelengths are not superimposed which is at variance with what is observed with complexes of proflavine with poly dAT and poly dG.dC. The beginning of the desorption process corresponds to minor variations at 445 nm, the maximum of absorption of the free dye, but large changes occur at 460 nm, the maximum of the difference spectrum of the complexes proflavine–poly dAT and proflavine-poly dG.dC. The spreading of the melting curves for different wave lengths must therefore reflect the dependence of the absorption spectra of the dye on the nature of the neighboring bases. However, the action spectrum of the fluorescence, which gives the absorption spectrum of the “emitting” sites only, is identical with the total absorption spectrum of the bound dye.  相似文献   

17.
W B Busa 《Cell calcium》1992,13(5):313-319
A systematic study of the spectral characteristics of the viscosity artifact in Fura-2 based [Ca2+] measurements reveals that, by selecting excitation wavelengths approximately 10 nm longer than those routinely employed and modestly reducing excitation bandpasses, the magnitude of the artifact can be reduced to experimentally undetectable levels without greatly impairing [Ca2+] measurements. The feasibility of this approach was confirmed on a ratio imaging microscope; the magnitude of the artifact observed in dextran-conjugated Fura-2 solutions prepared in water or in 50% sucrose was not statistically significant using an excitation wavelength pair of 361/389 nm, whereas at 350/380 nm [Ca2+] was underestimated by 34% in the higher viscosity solution. Thus, provided potential pitfalls are taken into account, a simple change in imaging protocol can avoid the viscosity artifact without recourse to correction factors. This approach may be employed either routinely, or else merely to test whether apparent [Ca2+]i differences observed at more conventional wavelengths arise from the viscosity artifact.  相似文献   

18.
Indo-1 is a fluorescent calcium probe used to measure intracellular free calcium concentrations. These measurements are often performed by comparing the fluorescence intensities of Indo-1-treated cells at two selected wavelengths corresponding to the maxima of the fluorescence spectra of the calcium-bound and calcium-free forms. In this study, we used an optical multichannel analyser to numerise the fluorescence emitted by a single cell. A computerised resolution of numerised spectra was used on intracellular Indo-1 fluorescence. Calculation of numerical and graphic estimators allows us to evaluate the fit of the resolution. Different sets of characteristic spectra were compared using this method. It appeared that no linear combination of the two known forms of Indo-1 and of the cell autofluorescence can fit with spectra of Indo-1-treated cells. In addition, a study of the physico-chemical properties of Indo-1 shows the existence of two other forms of the molecule: a protonated form (maximum emission at 455 nm) and a form in interaction with proteins (maximum emission at 438 nm). Taking into account the contribution of these two new forms leads to an improved spectral resolution of the fluorescence of Indo-1-treated living cells and, therefore, improves calcium measurements. Moreover, quantification of the amount of the protonated form of Indo-1 allows a measurement of intracellular pH at the same time as calcium determination.  相似文献   

19.
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.  相似文献   

20.
Mitochondrial damage is the main source of cellular injury upon ischemia–reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH.We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry.It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号