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

ANALYTICAL

BIOCHEMISTRY

193,

49-54

(1991)

SNARF-1 as an Intracellular pH Indicator in Laser Microspectrofluorometry: A Critical Assessment Olivier

Seksek,

Nelly

Henry-Toulmb,

Laboratoire de Physique et Chimie Biomoltkulaire, 4 Place Jussieu, Paris 75252, France

Received

October

Franck CNRS

Sureau,

UA 198, Universite’

Bolard

Pierre

et Marie

Curie,

18, 1990

The use of SNARF-l-AM (seminaphtorhodafluor-lacetoxymethylester) 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’C in the presence of 10 PM SNARF-I-AM, the internal concentration of SNARF-1 was approximately 200 j&M. 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 k 0.05), which corresponded with values obtained by conventional fluorometric methods. Q 1991 Academic PMS, IX

The measurement of the internal pH of cells by fluorometric methods has developed substantially in the past 10 years due to the appearance of pH-sensitive dyes and their acetoxymethylester derivatives (l-3). The latter allow rapid passive diffusion of the dyes across the 0003.2697/91 Copyright All rights

and Jacques

$3.00 Q 1991 by Academic Press, of reproduction in any form

plasma membrane of most cells. The esterified forms are hydrolyzed in the cytoplasm and the dye is trapped inside the cell. The determinations are generally made on a cell suspension with a conventional spectrophotometer. The mean value of intracellular pH is obtained over the cell population (e.g., (4-7)). However, it would be of more interest to measure this parameter on isolated cells in a heterogeneous population. Microspectrofluorometry, which is performed by coupling a microscope with a spectrofluorometer, enables the analysis of volumes as small as 10 pm3 and therefore the recording of fluorescence spectra of a single cell is possible (8,9). However, this technique requires information on the fluorescence emission spectrum. Until now, the available dyes had significant spectral changes in the excitation spectrum with two pH-sensitive bands. Recently, a new class of dyes, including SNARF-1,’ possessing two pHsensitive bands on the fluorescence emission spectrum was synthesized, allowing the study of intracellular pH by microspectroscopy. Here we report data, obtained by conventional spectrofluorometry and microspectrofluorometry, on the physicochemical conditions for the use of SNARF-1 with A20 lymphocyte cells. MATERIALS

AND

METHODS

Chemic& arzd reagents. SNARF-1 (seminaphtorhodafluor-1), BCECF (2’,7’-bis(carboxyethyl)-5(or 6)-carboxyfluorescein), and their acetoxymethylester derivatives (SNARF-l-AM and BCECF-AM) were purchased from Molecular Probes (Eugene, OR). Valinomycin, nigericin, digitonin, lipids, chicken egg white lysozyme, a-chymotrypsin, bovine serum albumin (BSA), and Hepes were obtained from Sigma Chemical Co. (St. ’ Abbreviations used: SNARF-1, seminaphtorhodafluor-1; BCECF, 2’,7’-bis(carboxyethyl)-5(or 6)-carboxyfluorescein; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; SUV, small unilamellar vesicle. 49

Inc. reserved.

50

SEKSEK

Louis, MO). Dimethyl sulfoxide (DMSO) was from Merck and of first grade purity. Calf thymus DNA, 1 mg/ml in 10 mM NaCl solution, was purchased from Pharmacia (Bois d’Arcy, France). Cell culture. A20 murine lymphocytes were a gift from Professor P. Sarthou (Departement d’Immunologie, Institut Pasteur, France) and cultured in RPM1 1640 medium (Bioproducts, MD) supplemented with 10% heat-inactivated fetal calf serum, 50 PM P-mercaptoethanol, 1 mM sodium pyruvate, 2 mM glutamine, and 50 U/ml penicillin-streptomycin (Flow Laboratories, Les Ulis, France) at pH 7.4. Cells were maintained at 37°C in a humidified atmosphere of 95% sir/5% CO, and were harvested when they reached a cell density of 2 X 106/ml. Cell viability was determined by trypan blue exclusion and was higher than 95% before each experiment. Loading with SNARF-I. Due to negatively charged carboxylic groups, SNARF-1 does not penetrate inside the cell. In contrast, the uncharged acetoxymethylester derivative (SNARF-l-AM) can passively diffuse across the plasma membrane to cytosol, where it is hydrolyzed by cellular esterases to free SNARF-1. It is this form which is fluorescent, and is consequently the pH probe. Cells were loaded with the fluorescent probe as follows: They were incubated 107/ml in supplemented RPM1 for 20 min at 37”C, 5% CO,, with 10 PM SNARF-l-AM from a 1 mM stock solution in DMSO. Control cells were tested with an equivalent amount of DMSO. Cells were then diluted lo-fold in supplemented RPM1 medium and incubated for 20 additional min at 37°C. They were centrifuged 10 min at 2OOg and the pellet was resuspended in a buffer containing 10 mM Hepes, 145 mM NaCl, 5 mrvi KCl, 5 mM dextrose, 1 mM CaCl,, 1 mM KH,PO,, 0.5 mM MgSO, adjusted to pH 7.4 (Hepes saline buffer). Cells were washed once more, and the pellet was kept at room temperature until use. Intracellular pH calibration. In vivo pH calibration was performed according to the method developed by Thomas et al. (1). Briefly, after incubation with the fluorescent probe, cells were washed in a buffer containing 10 mM Hepes, 130 mM KCl, 20 mM NaCl, 5 mM dextrose, 1 mM CaCl,, 1 mM KH,PO,, 0.5 mM MgSO, at various pH values obtained by addition of small amounts of 0.1 M solutions of KOH and HCl. The pH changes of the external buffer of the cell suspension were followed with a Tacussel Isis 20000 pH-meter. Addition of nigericin (1 pg/ml) and valinomycin (5 pM) allowed an exchange of K+ for H+ which resulted in a rapid equilibration of external and internal pH. In the pH range studied (5.8-8.5), cell viability was not significatively affected. Microspectrofluorometer. Emission spectra on a single living cell were made with an uv-visible microspectrofluorometer prototype developed in our laboratory, which will be extensively described elsewhere (8,9).

ET AL.

Briefly, excitation was achieved by an argon laser beam, using the 514.5-nm line. A 100X objective and a luminous field diaphragm were used on the excitation path to produce a circular illuminated area of l-2-pm in diameter; thus only a small area inside one cell was irradiated. Fluorescence spectra were recorded in the region 520-780 nm on a 1024 diode intensified optical multichannel analyzer (Princeton Instruments). To allow cell survival and to avoid photochemical effects, excitation beam power was minimized by the use of neutral density filters. Typically, the power was reduced to 0.1 PW for the illuminated circular area used, and, under these conditions, it was not possible to detect a decrease in fluorescence intensity or spectrum change during the short periods of illumination (l-5 s) used for experiments. Data were stored and processed on a 80286 IBM PSI2 microcomputer using the Jobin-Yvon “Enhanced Prism” software. Spectrofiuorometric measurements. Fluorescence emission spectra of SNARF-1 solutions and SNARF-lAM-loaded cell suspensions were recorded in a JY 3D spectrofluorometer (Jobin-Yvon, France) equipped with a thermostat set at 25°C and a stirred cell compartment. The fluorescence of the probe was excited at 514 nm, while the emission was recorded in the range 540-770 nm. Preparation of small unilamellar vesicles (SUV). SUV were prepared according to Newman and Huang (10). Briefly, vesicle suspensions were prepared by dissolving known amounts of phospholipids in chloroform. After removal of the solvent, 2 ml of phosphate buffer containing 7 mM Na,HPO,, 3 mM KH,PO,, and 150 mM NaCl, pH 7.4, was added. The sample was sonicated above the transition temperature of pure phospholipid vesicles for 20-30 min under nitrogen. RESULTS

The fluorescence emission spectra of SNARF-1, obtained in Hepes saline buffer, are shown in Fig. la. Two bands located at 635 and 590 nm are present. The ratio (R) of the intensities of these bands was clearly dependent on the pH of the buffer (Fig. lb). An isobestic point at 610 nm indicated the existence of two forms of SNARF-1 at equilibrium. Control experiments showed that the fluorescence spectrum of this molecule is sensitive neither to ionic strength changes (for concentrations ranging from 100 to 300 mM in Na+ and K+) nor to Ca2+ concentration. In contrast, the quantum yield of the probe and R were highly sensitive to temperature changes: the fluorescence intensity dropped by 25% when the temperature shifted from 25 to 37°C. At the same time, R increased from 1.7 to 1.85 at pH 7.4. The studies described above were done using a classical spectrofluorometer with quartz curvettes. Similar results were obtained when experiments were performed with the microspectrofluorometer, once the apparatus response had been taken into account. The influence of

SNARF-1

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FIG. 1. Effect of pH on the fluorescence emission spectra of SNARF-1 fluorometry with an excitation wavelength of 514.5 nm (F in arbitrary cence emission intensity at 590 nm) plotted versus pH.

(1 pM) in Hepes saline buffer. (a) Spectra obtained by microspectrounits). (b) R = (fluorescence emission intensity at 635 nm)/(Auores-

the probe concentration on the value of R was also analyzed by microspectrofluorometry. A very low working volume minimized sample absorbance, and consequently inner filter effect. This allowed us to check that variation of SNARF-1 concentration had no influence on R for values ranging from 1 to 500 PM. Once the parameters of the probe were determined in Hepes saline buffer, we examined by microspectrofluorometry the behavior of this molecule inside the cell. The cells were loaded on the microscope slide during the recording of the fluorescence spectra. In Fig. 2, both the fluorescence intensity of the cytoplasm and R are plotted as a function of the incubation time. The fluores-

cence intensity of the probe inside the cell reached a steady level after 20 min. Additionally, R, which is theoretically a reff ection of the pH, varied as the concentration of the probe increased in the cell. This variation is discussed later. On the other hand, an experiment was designed to determine the concentration of the probe inside the cell, once the fluorescent plateau was reached. The loaded cells were then pelleted, resuspended in Hepes saline buffer, and permeabilized with 100 PM digitonin. SNARF-1 concentration was determined by conventional spectrofluorometry by comparing the fluorescence spectra of the permeabilized cell suspensions with the spectra recorded on solutions of known concentrations. The obtained value was then related to the cellular volume of the suspension. This volume was estimated by assuming cells were 15-pm-diameter spheres. Consequently, when cells were incubated with 10 pM SNARF-l-AM, the intracellular concentration was calculated as 200 -+ 50 ELM. This was an approximate 20-fold accumulation of the probe into the cell. This is a classical result for esterified probes. More surprising was the fact that the addition of digitonin resulted in a 3-fold increase in fluorescence. This indicated that the fluorescence of the probe was quenched inside the cell. We then determined the rate of leakage of the probe out of the cells. Cells were centrifuged at various time intervals after the end of the loading period, and the fluorescence signal in the supernatant was measured by conventional spectrofluorometry. Figure 3 shows that considerable fluorescence rapidly appeared in the supernatant, indicating a significant leakage of the probe from the cells. This implied that no accurate measurement of the intracellular pH could be done on a cell suspension by conventional spectrofluorometry.

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time (mink FIG. 2. Fluorescence intensity (8’) of SNARF-1 (0) and corresponding ratio R (W) detected by microspectrofluorometry on one living cell, as a function of the time following addition of 10 pM SNARFl-AM to external RPM1 medium (pH 7.4) at 21°C (F in arbitrary units).

52

SEKSEK

120 A) *y

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FIG. 3. Extracellular leakage of the probe: The results are expressed as the ratio of the fluorescence coming out of the supernatant to the fluorescence of the cell suspension prior to centrifugation. Values are given as percentages. Measurements were done at different time intervals after the loading of SNARF-l-AM.

An in vivo calibration curve to establish the relationship between R and the intracellular pH was calculated by using the ionophores nigericin and valinomycin to impose cytoplasmic pH on A20 cells as described under Materials and Methods. The results presented in Fig. 4 raised unexpected points. Although the profile of the curve of the ratio R, as a function of the pH obtained in the cells, was similar to the Hepes buffer curve (Fig. la), the value of R for a given pH was substantially different. More information was necessary to understand this difference. SNARF-1 displayed two significantly different characteristics inside the cells compared to buffer alone:

F

a

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quenched fluorescence and reduced R values. Therefore, the effects of the major cellular macromolecules on probe fluorescence properties were studied. First, DNA was examined. The fluorescence of a 1 ELM solution of SNARF-1 in Hepes saline buffer and increasing concentrations of double-stranded DNA (100 j.&M to 2 mM) was measured. No modification of SNARF-1 fluorescence nor of DNA absorbance was observed. The same experiment was performed with heat-denatured DNA and no changes in SNARF-1 fluorescence resulted. We concluded that the fluorescence changes observed inside the cells did not result from an interaction of the probe with DNA. Second, the interaction with membranes was investigated using small unilamellar vesicles as model membranes. Two types of lipid composition were tested: one containing only neutral phospholipids and the other one containing neutral and negatively charged phospholipids in the ratio 8:2. In both cases, total phospholipid concentration was varied from 1 to 5 mM without inducing any change in the fluorescence of SNARF-1. Finally, BSA, a-chymotrypsin, and lysozyme were used to examine the possible interaction of SNARF-1 with proteins. Figure 5 shows a reduction in both fluorescence intensity and R in the presence of BSA. However, with lysozyme, only a small decrease in the fluorescence was observed, with no significant change in R. After addition of cu-chymotrypsin, a small decrease in R was observed, but no fluorescence quenching occurred. Only the interaction with BSA led to a significant decrease in the ratio R, which was also observed in cells. The decrease in the value R could also originate in the storage of a part of the probe in acidic compartments such as lysosomes, which could be insensitive to the pH

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FIG. 4. Microspectrofluorometric fluorescence emission spectra of SNARF-l-AM-loaded A20 lymphocytes. (a) Emission spectra. Calibration of intracellular pH was obtained using nigericin (1 rg/ml) and valinomycin (5 PM) in Hepes buffer containing 130 mM KC1 (Fin arbitrary units). (b) R = (fluorescence emission intensity at 635 nm)/(fluorescence emission intensity at 590 nm) plotted versus pH. For each pH value, number of cells = 5.

SNARF-1

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FIG. 5. Effect of proteins on SNARF-1 fluorescence. (a) Effect of protein concentration on the fluorescence emission intensity at the isobestic point (610 nm) of 1 pM SNARF-1 in Hepes saline buffer. Excitation wavelength = 514 nm. T = 25°C. BSA (0); lysozyme (X) (Fin arbitrary units). (b) Effect of proteins on pH calibration with 1 FM SNARF-1. Concentration of BSA (0) and lysozyme (X): 10 mg/ml (control without protein (0)).

changes induced during the calibration procedure. This question was addressed by sequential opening of the various compartments with a graded series of detergents as previously described by Nieminen et al. in rat hepatocytes (11): 10 PM digitonin opens cytosol, and 50organelles. During the 100 PM opens nonmitochondrial sequential opening of compartments by the action of the detergents on a suspension of cells loaded with SNARF-l-AM, the fluorescence intensity was recorded. After the addition of 10 pM digitonin, which opened the cytosolic compartment, an increase in the signal was observed. This was thought to be due to the dilution and the subsequent dissociation of the protein-associatedSNARF-1 complex in the buffer. Higher concentrations of digitonin, up to 100 PM, had no further effect, implying that no significant compartmentation of the probe into acidic organelles occurred. We experimentally dein 84 exponentially growing termined R using SNARF-1 cells, and then calculated pH, from the previously plotted calibration curve. Results are shown in Fig. 6. The mean was 7.18 f 0.10 at an external pH of 7.40 f 0.05. This value was confirmed by the experiments performed with BCECF and spectrofluorometry on a population of cells by classical method. In fact, we obtained 7.19 f 0.12 (8 measurements) for the same external pH. DISCUSSION The choice of a probe for measuring intracellular ion concentration is influenced by the characteristics of the probe-ion interaction and also by the behavior of the probe inside the cell. A number of new probes are now available to measure intracellular pH, but little is known about their biological behavior. We report here the characterization of one of these new probes,

SNARF-1, which can be used in microspectrofluorometry. Indeed, compared to the extensively used BCECF (e.g., (2,4-7,12-17)) which presents a pH-sensitive fluorescence excitation spectrum, SNARF-1 presents a two-band, pH-sensitive emission spectrum. This property allows direct determination of the ratio related to the pH, using only one excitation wavelength. In contrast, with BCECF in digital imaging or microfluorometry, it is necessary to excite the sample sequentially at two different excitation wavelengths to obtain a fluorescence ratio (12-17) or to estimate pH from the fluorescence intensity excited at only one wavelength (l&19). Additionally, SNARF-1 is excited at 514 nm, which is a laser line less absorbed by the cell than a near-uv one; for example, another probe with two pH-

10 number of cells

8 6 4 2 0

FIG. 6. Distribution Hepes saline buffer

of R and pH, values at pH 7.4 (n = 84).

in a A20 cell population

in

54

SEKSEK

sensitive bands on the fluorescence emission spectrum, 2,3dicyanohydroquinone, is excited at 360 nm (20). In solution, it appeared that SNARF-1 fluorescence is not modified by the variation in the ionic strength or the nature of the buffer. However, fluorescence intensity and band ratio were dependent upon temperature. With cells, several problems occurred. First, the probe leaked rapidly, inducing a prevailing contribution of the external probe to the total fluorescence intensity since the fluorescence was quenched inside the cells. Therefore, accurate measurement of pH, with SNARF1 by classical spectrofluorometry was not possible. However, because fluorescence of the external medium negligibly contributed to the total signal in microspectroscopy, we have been able to bypass this problem. Second, determination of the absolute pH values required preliminary calibrations for each studied state. Attention must be paid to the fluorescence changes occurring in the cells compared to the buffer alone. These variations could arise from an acidification of the cytoplasm originating from the hydrolysis of the esterified probe, from the localization of the probe in acidic compartments of the cells, or from its interaction with intracellular components. The first explanation can be discarded because of the high buffering capacity of the cytoplasm. If a buffering power of -50 mM/pH for mammalian cells is assumed (21), N 15 mM intracellular protons would be necessary to explain the 0.3-pH unit difference corresponding to the observed decrease in fluorescence. The concentration of SNARF-1 present inside the cell was not compatible with these values. The second explanation is eliminated by our experimental results. Indeed, no significant localization of the probe in the acidic compartment could be detected. Finally, it is more likely that the interaction of the probe with intracellular proteins was responsible for the decrease of R independent of cytoplasmic pH changes. Regardless, we observed that when the level of SNARF-1 inside the cell was stabilized, R also reached a steady level, which probably corresponds to the equilibrium of the probe between its different targets. It is important to keep this point in mind when the purpose is to detect the pH changes induced by biological or chemical effecters. These molecules may disturb the equilibrium of the probe distribution and consequently induce fluorescence changes which would not be related to pH changes. We have seen that with A20, the mean cellular value was 7.18 f 0.10 at an external pH of 7.40 + 0.05. This value correlates well with the values obtained in the literature for lymphocytes (e.g., (3-5,21-24)). In conclusion, the probe can be confidently used to measure intracellular pH by microspectrofluorometry, provided that certain conditions are fulfilled: working at biologi-

ET

AL.

cal equilibrium studied state.

and performing

a calibration

for each

ACKNOWLEDGMENT This France).

research

was supported

in part

by the Institut

Curie

(Paris,

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