Thyrotropin-releasing hormone-induced changes in intracellular [Ca2+] measured by microspectrofluorometry on individual quin2-loaded cells

We have developed an accurate and practical method for measuring intracellular Ca2+ concentration [( Ca2+]i) in single cells in monolayer culture using the fluorescent Ca2+-binding dye quin2. Quin2 was loaded into cells as a membrane-permeant ester which is hydrolyzed in the cytoplasm to the impermeant free acid, which is the indicator form (Tsien, R.Y., T. Pozzan, and T.J. Rink, 1982, J. Cell Biol., 94:325-334). The method involves the measurement of fluorescence at 340- nm excitation (I340), where dye fluorescence is dependent on Ca2+, and at 360-nm excitation (I360), where dye fluorescence is independent of Ca2+. The ratio of these two values (I340/I360) is thus related to the concentration of Ca2+ but independent of dye concentration and can be used as a measure of [Ca2+]. To test the ratio method in the microscope, we measured [Ca2+]i in GH3 cells in monolayer culture. We found a resting [Ca2+]i of 44 +/- 28 nM (mean +/- SD, n = 34), as compared with a suspension value (Gershengorn, M., and C. Thaw, 1983, Endocrinology, 113:1522-1524) of 118 +/- 18 nM. We also measured [Ca2+]i during stimulation of the cells with thyrotropin-releasing hormone (TRH) and found a 2.4-fold increase above resting levels within 20 s, a trough at 73% of resting at 90-100 s, and a peak slightly above resting at 3 min. Depolarization of the plasma membrane with KCl produced a sustained increase in [Ca2+]i. All of these data are in good agreement with the results of Gershengorn and Thaw on suspension cultures. When measuring both resting [Ca2+]i and the effects of TRH and KCl on small groups of cells, we found some variation among experiments. Using an image intensifier-video camera, we videotaped cells during TRH stimulation. Digital image analysis of these pictures demonstrated that there was a large variation in responsiveness from cell to cell. The microscope ratio method offers the possibility of resolving regions of differing [Ca2+] within the cytoplasm.     

Dicarboxy-dichlorofluorescein: a new fluorescent probe for measuring acidic intracellular pH

Derivatives of fluorescein sensitive to pH are extensively utilized for the determination of intracellular pH (pHi). Available dyes have pKa values of approximately 7.0, and are not well suited for measuring acidic pHi. We examined the fluorescein derivative, 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDCF) for its potential in the microspectrofluorometric measurement of pHi during acidic conditions. CDCF showed intense fluorescence and pH sensitivity near its "effective" pKa value of 4.2, using a 495/440 nm dual excitation wave-length ratio method. Protein interactions caused fluorescence ratio deviations which were most pronounced at the extremes of pH, whereas calcium and magnesium concentrations had little effect on the fluorescent ratio intensity. Intracellular calibration performed using nigericin in the presence of high potassium eliminated the need to correct for protein interactions, and the ratio method minimized any variations due to dye concentration differences or instrument fluctuation. Intracellular retention of the dye was high, and 95% of the initial signal remained after 1 h. Fluorescence bleaching was 14.5% after 1 h of continuous excitation and cell survival was not affected by dye loading. We conclude that CDCF is an excellent intracellular pH indicator in the pH range of 4-5.     

Role of calcium in differentiation of murine erythroleu-kemia cells

Calcium plays a crucial role in the normal and abnomal cell metabolism.The role of calcium in the differentiation process of murine erythroleukemia cells(MELC)remains controversial.Here,based upon quantitative measurement of fluorescence in single cells,a method was developed to investigate the intracellular free calcium[Ca^2 ]i concentration and DNA contents simultaneously,by employing the fluorescent probe,fluo-3 acetoxymethyl ester and DNA dye Hoechst 33342.During MELC differentiation.[Ca^2 ]i concentration incresed.We also demonstrated that calcium ionophore,A23187,enhanced the HMB-induced MELC differentiation,while verapamil,an inhibitor of calcuim uptake,slightly reduced differentiation.These results suggested that an increase in the [Ca^2 ]i level was an essential step in HMBA-induced MELC differentiation.     

Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.     

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.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.     

Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells

The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.     

Improved measurements of intracellular nitric oxide in intact microvessels using 4,5-diaminofluorescein diacetate

4,5-Diaminofluorescein diacetate (DAF-2 DA) has been widely used for the measurement of nitric oxide (NO) in living cells and tissues. We previously established a method that demonstrated platelet activating factor (PAF)-induced endothelial NO production in intact venules using DAF-2 DA. In previous applications, the loading dye was removed from the extracellular space before NO measurements. However, in high permeability vessels, endothelial cells quickly released the accumulated intracellular DAF-2 after the washout, which compromises the NO measurement. The objective of this study was to investigate if the presence of DAF-2 DA during NO measurements could overcome the dye retention problem and enhance the sensitivity of NO detection. Experiments were conducted in individually perfused rat venules, and endothelial NO was measured using fluorescence imaging under basal and stimulated conditions with continuous perfusion of DAF-2 DA. Continuous dye perfusion was found to promote a relatively constant endothelial dye concentration in both normal and high permeability vessels throughout the experiment. With the use of this method, the basal and stimulated NO was quantified after endothelial DAF-2 concentrations reached a steady state. Our results showed enhanced sensitivity of detecting PAF-stimulated NO compared with a previous method. We also found that the hydrolyzed intracellular DAF-2, the precursor of DAF-2 triazole, contributed significantly to the measured fluorescence and that an appropriate subtraction of non-NO-dependent intracellular DAF-2 fluorescence is critical for the assessment of NO in living tissues. This method overcame the dye leakage problem, enhanced the sensitivity of NO detection, and improved NO quantification, demonstrating significant advantages over existing methodologies using DAF-2.     

Abnormal spectra alteration observed in Triton calibration method for measuring [Ca2+]i with fluorescence indicator, fura-2

We compared two commonly used calibration methods for measuring the concentration of intracellular free calcium ([Ca2+]i) by ratiometric fluorescence dye, fura-2 in mouse neuroblastoma-rat glioma hybrid cells (NG108-15). One calibration method, the Triton method, employs detergent Triton X-100, while the other, the Ionomycin method, uses a calcium-specific ionophore, Ionomycin. In the Triton method, we observed that at excitation 380 nm, the fura-2 fluorescence intensity of steady-state cells abnormally situated beyond the limiting intensity for calibration. By excitation scan, we demonstrated that this abnormality was caused by the change of fura-2 isosbestic points, which in turn was due to cell lysis after the addition of Triton X-100. This problem was resolved in the Ionomycin method by avoidance of cell lysis. Our results showed the correlation between inconsistent isosbestic points and cell lysis. As the basis for [Ca2+]i calibration, the proportionality between the fluorescence intensity and the concentration of dye species was impaired because of inconsistent isosbestic points. This inconsistency can be eliminated by a preliminary experiment of excitation scan to test the feasibility of different calibration methods.     

Non-linear relationship between fluorescence and membrane potential

The fluorescence intensity of the cynanine dye DiO-C₆-(3)_in 0.32% suspension of Ehrlich ascites tumor cells was determined under various potassium concentrations. In addition, the fluorescence levels of cell-free buffer solutions and those of supernatants were measured. For every potassium concentration the partition of the dye between cells and medium was calculated and its relation to the potassium gradient was given. A model was developed which assumes the fluorescence of a cell suspension to be the sum of the fluorescence signal of dye in the extracellular medium and that of cell-associated dye. A calibration curve of fluorescence vs. membrane potential was constructed. Neither the fluorescence of the cell suspension nor that of the supernatants was a linear function of the membrane potential. The limitations of membrane potential determination by fluorimetric methods are discussed.     

Intracellular alkalinisation in Vero cells parasitised by Trypanosoma cruzi

We studied the intracellular pH of Vero cells parasitised by Trypanosoma cruzi, using different methods: fluorimetric measurement after labelling the cells with the pH-sensitive intracellular fluorescent dye 2′,7′,-bis- (2-carboxyethyl)-5- (and-6)-carboxyfluorescein, acetoxymethyl ester; flow cytometry; and image analysis after staining the cells with neutral-red vital stain. The results show that the intracellular pH of the parasitised cells rose in comparison with that of the uninfected control cells. A study of the population of parasitised cells made by flow cytometry allowed us to subdivide the cells from the infected cultures into two populations according to their pH as obtained by fluorimetric measurements. Image analysis showed that the cell cytoplasm was more alkaline in the vicinity of the sites containing parasites. Treatment of the parasitised cells with amiloride, ouabain, or with 4,4′-diisothiocyano-2,2′-stilbene disulphate consistently lowered the pH values of the parasitised cells, but not sufficiently to return to the values of the non-parasitised control cells. When the control cells were subject to similar treatments with the inhibitors, only amiloride acidified the cytoplasm to any extent. The basification undergone by the parasitised cells was independent of the transport systems and may be a consequence of the release of NH⁺₄ by the intracellular amastigotes. © 1998 Australian Society for Parasitology.     

Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine

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.     

Assessment of monocyte esterase activity by flow cytophotometry.

An azo dye supravital method has been devised for selectively staining human monocytes in suspension for nonspecific esterase activity. Stained cells can be identified and rapidly enumerated by presenting the suspension of stained cells to the Cytograf, a flow-through cell discriminating cytophotometer. The intensity of stain is proportional to the intracellular esterase activity. By analysis of the oscilloscope display, it has been possible to obtain relative data concerning the degree of activity of monocyte nonspecific esterase activity. These observations suggest a unique approach to the measurement of intracellular enzyme activity in selected cells in a mixed population.     

Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader

Microscopic counting of plant cells is a very tedious and time-consuming process and is therefore seldom used to evaluate plant cell number on a routine basis. This study describes a fast and simple method to evaluate cell concentration in a plant cell suspension using a fluorescence microplate reader. Eschscholtzia californica cells were fixed in a mix of methanol and acetic acid (3:1) and stained with a fluorescent DNA binding dye (Hoechst 33258). Readings were done in a fluorescence microplate reader at 360/465 nm. Specific binding of the dye to double-stranded DNA was significantly favored over unspecific binding when 1.0 M Tris buffer at pH 7.5 containing 1.0 M NaCl and 75 microg ml(-1) of Hoechst 33258 was used. Fluorescence readings must be done between 4 min and 12 min following the addition of the staining solution to the sample. The microplate counting method provides a convenient, rapid and sensitive procedure for determining the cell concentration in plant cell suspensions. The assay has a linear detection range from 0.2 x 10(6) cells to 10.0 x 10(6) cells per milliliter (actual concentration in the tested cell suspension). The time needed to perform the microplate counting was 10% of that needed for the microscopic enumeration. However, this microplate counting method can only be used on genetically stable cell lines and on asynchronous cell suspensions.     

Yeast vitality determination based on intracellular NAD(P)H fluorescence measurement during aerobic-anaerobic transition

This work presents a yeast-cell vitality-assessment method based on on-line intracellular fluorescence measurement. The intracellular NAD(P)H fluorescence of a cell suspension is recorded during transition from aerobic to anaerobic conditions and the output signal is evaluated as a measure of yeast vitality (quality). This fluorescence method showed a highly satisfactory correlation with even low dead cell numbers where the acidification power test could not be applied.     

SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment

The use of SNARF-1-AM (seminaphtorhodafluor-1-acetoxymethylester) to measure the internal pH of a single living cell by laser microspectrofluorometry has been analyzed with a lymphocyte murine B cell line A20. After incubation of the cells at 37 degrees C in the presence of 10 microM SNARF-1-AM, the internal concentration of SNARF-1 was approximately 200 microM. The enhancement of fluorescent intensity of the probe is concomitant with its leakage out of the cells. During the measurement period, this induces a continuous increase of the contribution of the external probe to the total fluorescence intensity. This prevented classical spectrofluorometry measurements, but did not preclude microspectrofluorometry measurements of internal pH. The ratio R was calculated from fluorescence intensities at 635 and 590 nm and used as an indicator of the intracellular pH. Calibration curves of the intracellular pH were obtained in the presence of nigericin and valinomycin. It appeared that both the fluorescence intensity and the ratio R were lower inside the cell than those values obtained in aqueous solutions. Possible interactions with the main biological macromolecules (i.e., DNA, proteins, membranes) were investigated as well as a possible compartmentation of the probe in cellular organelles. The modifications of probe characteristics inside the cells were attributed to the binding of the probe to cellular proteins. The intracellular pH of A20 cells, measured by SNARF-1 on 84 cells, was found to be 7.18 +/- 0.10 (with an external pH of 7.40 +/- 0.05), which corresponded with values obtained by conventional fluorometric methods.     

Comparison of spectrum-shifting intracellular pH probes 5'(and 6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1'(3'H)-isobenzofuran]-3'-one and 2',7'-biscarboxyethyl-5(and 6)-carboxyfluorescein.

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.     

Simultaneous determination of intracellular calcium concentration and histamine secretion in rat basophilic leukemia cells (RBL-2H3).

In rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mast cells, the aggregation of IgE receptors initiates increase in the intracellular concentration of calcium ([Ca2+]i), monitored with the fluorescent Ca probe fura-2, and finally results in histamine secretion. In cell suspensions, however, the fluorescence gradually increases due to leakage and exocytosis of the dye. A superfusion system was developed to overcome these problems and [Ca2+]i was calculated from the ratio of fluorescence intensities at 505 nm of fura-2 excited at 340 and 380 nm. Histamine and beta-N-acetylglucosaminidase in granules are released during exocytosis, and both substances in the superfusates were determined simultaneously. This system is useful for studies on the relationships of cell stimulation, changes in second messengers, and final responses.     

Improved method for measuring intracellular Ca++ with fluo-3

The accuracy of flow cytometric measurement of intracellular calcium with fluo-3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo-3 and SNARF-1. When SNARF-1 fluorescence is collected at approximately 600 nm, its intensity does not change upon cell activation. Furthermore, fluo-3 and SNARF-1 fluorescence signals exhibit a linear relationship. The ratio of fluo-3 to SNARF-1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo-3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+]i.