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Quin-2 and Fura-2 measure calcium differently
186,28-30
ANALYTICALBIOCHEMISTRY
Quin-2 and FuraD. L. Mazorow2
(1990)
Measure Calcium Differently’
and D. B. Millar*
Immunobiology and Transplantation Department, Naval Medical Research Institute, Bethesda, Maryland 20814, and *Laboratory of Molecular Genetics, National Institutes of Mental Health, St. Elizabeth’s Hospital, Washington, DC 20032
Received
July
24,1989
Several fluorescent probes have been used in the past to monitor and to measure intracellular calcium and calcium fluxes. The most widely used of these probes are those developed by Tsien. We address the markedly different values obtained when comparing Quin-2 (the original probe) with Fura(a second-generation probe). In most cases the values for intracellular calcium have been considered to be interchangeable for the different probes. Using several different hematopoietic cell lines we show that in no case do the two o 1990 Academic PRSS, IIIC. probes yield equivalent values.
The detection and measurement of intracellular calcium is of the upmost importance to understanding cell function. To date the most consistent information has been obtained from a variety of fluorescent probes. For years the arsenazo dyes (1) as well as the photoproteins obelin (2) and aequorin (3) have been used to monitor changes in intracellular calcium. The disadvantage of these probes is that the cell needs to be permeabilized to allow entry of the probe. However, in 1980 Tsien (4) described a new fluorescent probe used to monitor intracellular calcium events. This new probe was called Quin2. The beauty of this technique was that the probe was available as a cell-permeable ester (Quin-2 AM). Once inside the cell, cytosolic esterases would deesterify the Quin-2 AM into Quin-2 which is not readily membrane permeable. Thus, the probe was placed in the cytosol without undue trauma to the cell membrane. However, Quin-2
has
a relatively
high
affinity
for
calcium
which
has generated concern that Quin-2 may strip calcium 1 The opinions here are the sole responsibility of the authors and in no way constitute official U.S. Navy policy. This work was supported by Navy Research and Development Command Work Unit MRO 4120-06002. * Present address: Environmental Medicine Department, Naval Medical Research Institute, Bethesda, MD 20814. To whom correspondence should be addressed. 28
from cell stores, yielding misleadingly high values for cytosolic calcium. Additionally Quin-2 bleaches appreciably over time which increases the difficulty in obtaining accurate values for F,,,,, and Fmi,. The advantage of Quin-2 over the other fluorescent probes mentioned is the ease of entry into the cell relative to the other calcium-sensitive probes described above. A few years later Tsien’s lab developed several other calcium-sensitive fluorescent probes which had improved capabilities compared to the Quin-2 probe (5). These were called Indo-l and Fura-2. These probes had a higher quantum yield than Quin-2, did not bleach as readily, and were more easily calibrated to “actual” calcium concentrations. Also, these probes had a more specific affinity and a lower binding constant than Quin-2 for calcium. We present here a comparison of Quin-2 and Fura- measurements of cytosolic calcium which suggest that the concerns over Quin-2 may be justified. METHODS
AND
MATERIALS
Neutrophils were isolated by standard procedures (6). Tissue culture cells (Jurkat, a human T-cell lymphoma cell line, K562, KGl, and KGla human myeloid leukemia cell lines) were grown in RPM1 1640 (Hazelton), 10% fetal calf serum (Hazelton), 2 mM glutamine (Cellgro), and penicillin (50 III/ml)-streptomycin (50 pg/ml) (Cellgro). The cells were incubated with either Quin-2 AM (Calbiochem) or Fura- AM (Calbiochem) for 30 min in a CO, incubator (37°C). Cells were centrifuged and resuspended to a final concentration of 5 X lo5 cells/ ml in Hanks’ balanced salt solution (without phenol red) with 15 mM Hepes.3 Resting calcium levels with Quin-2 were determined by the technique described by Grynkiewicz et al. (5). Briefly, [Cal = Kd(F - Fmi,)/(Fmax - F). The dissociation constant Kd = 115 nM, F is the measured fluorescence, and Fmin and F,,, are the minimum 3 Abbreviations used: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, EGTA, ethylene glycol bis(j3-aminoethyl N,N’-tetraacetic acid. 0003-2697/90
Copyright All rights
0 1990 by Academic
of reproduction
in any form
ether)$3.00
Press, Inc. reserved.
CALCIUM
MEASUREMENT
WITH
QUIN-2
The dissociation constant Kd = 135 nM, R is the measured ratio, Rmin and R,,, are the minimum and the maximum ratios obtained using Fura(acid form) in salts without calcium (10 mM EGTA) and with calcium (1 mM), Sfl is the measurement of free dye at the second excitation wavelength (in low-calcium salts), and Sb2 is the measurement of bound dye (in high-calcium salts) at the second excitation wavelength. The second technique is a direct calibration of fluorescence intensity using solutions of known calcium concentration made from mixtures of calcium salt and EGTA according to the stability constants of Martell and Smith (8) at a known pH, temperature, and ionic strength (5). The calibration also has an adjustment for pH, temperature (using the Van% Hoff equation), and ionic strength (values published by Tsien). The two methods of determining calcium concentration with Furaagreed within error, and our values agreed with those published by Grynkiewicz et al. (5) within 5 nM. Each calcium concentration was determined from at least five separate samples and the standard deviations and error were determined. Fluorescence measurements were obtained in a Perkin-Elmer Model 44B spectrophotofluorometer equipped with stirring and temperature control. All values obtained were at 37°C unless otherwise indicated. Quin-2 measurements were made with an excitation wavelength of 339 nm (slit 4 nm) and emission was determined at 492 nm (slit 6 nm). The Furastudies used excitation wavelengths of 335 and 362 nm (slit 4 nm) and emission was monitored at 510 nm (slit 6 nm). To check the accuracy of the system relative to the published values of Tsien, Furameasurements were also obtained at excitation wavelengths of 340 and 380 nm with emission set at 510 nm. Our values were in excellent agreement with those of Grynkiewicz et al. (5). RESULTS
In no case did measurements of Quin-2 and Furagive equal results. The most dissimilar of the results is the variance between Quin-2 and Furain neutrophils. The literature on neutrophil intracellular calcium levels using Quin-2 abound with values varying from 100 up to 200 nM (9-13). Our calcium values for neutrophils using
29
FURA-P TABLE
and maximum fluorescence, respectively. F,,, is obtained by sonically lysing the cells and releasing the intracellular Quin-2. Fmin is obtained by adding EGTA (10 mM final) (Fluka puriss) to the sonically disrupted solution and adjusting fluorescence intensity for change in volume. Calcium concentrations with Furawere determined using two somewhat independent means. The first is described by Tsien et al. (7) and consists of the equation
[Cal = & [(R - Rrnin)I(Rmax - ~)1(&2/&~2)-
AND
1
Comparison of Cellular Resting Calcium between Quin-2 and FuraCa concentration
(nM)
FuraCell type Neutrophil K562 Cadaveric Jurkats KG1 KGla
Quin-2
bone marrow
’ Intracellular was determined sity and solutions tures of calcium * Intracellular by Grynkiewicz
160 63f 72 102 95 73
+ 70 5 k 20 f 14 + 30 + 25
Standard
curve”
16f 3 50 f 10 36t 4 60f 5 32f 3 29+ 2
Equationb 14 55 43 54 37 35
f t i k + +
3 8 4 5 6 7
calcium was determined using a standard curve which using a direct calibration of Furafluorescence intenof known calcium concentration made from mixsalt and EGTA. calcium was determined using the equation described et al. (5) with R, &in, R,,,, Sn/Sb:!, and Kd = 135 nM.
Quin-2 were between 90 and 230 nM. However, we are not the only ones who obtained a very low value for resting calcium in neutrophils using Fura-2. Perez et al. (14) found resting values of calcium at 14 nM. Using either technique of Fura- analysis, the values we obtained for resting calcium were quite low. The resting calcium was between 11 and 19 nM. Our values were obtained from 12 different individuals. We also tested several other hematopoietic cells and cell lines (see Table 1). In Table 1 we compare the resting calcium levels of Quin-2 and Fura-2. In every case Quin-2 fluorescence yielded values which indicated higher cytosolic calcium levels than Fura-2. In all cases except K562 cells, the levels of cytosolic calcium using Quin-2 were at least twice the levels of Fura-2. DISCUSSION Although for many studies it is not essential to know the absolute calcium concentration within a cell, but to monitor changes in the cytosol after treatment with a ligand, it is important to note that equivalent values are not obtained with the different calcium probes, We wish to show not only that the indicated resting calcium levels of various hematopoietic cells differ among each other, but also that dissimilar values are obtained when using Quin-2 or Fura-2. There are many possible reasons for this. To measure Quin-2 one assumes that the pH and ionic strength within and without the cell are the same (which is not true). The other fact that needs to be remembered is that Quin-2’s affinity for calcium is within the same magnitude as that of many biological functions for calcium. Therefore, Quin-2 may shift the equilibrium of the cytosol as well as strip cytosolic stores of bound
30
MAZOROW
calcium. In the case of the neutrophil, Quin-2 may be pulling calcium from the granules as well as from other places. For the primitive relatively undifferentiated K562 cell line, the agreement between Quin-2 and Fura2 may reflect less complex modes of calcium storage. In conclusion, if one is only interested in a relative increase or decrease in cytosolic calcium, either of the probes should prove to be adequate, although Fura- will be more sensitive to small changes. However, if one is trying to establish true “values” for cytosolic calcium, then Fura- is a better choice. Also, Fura- is less toxic to the cell and may give a picture of events which more closely resembles true conditions. ACKNOWLEDGMENTS We thank Dr. Carl June for the kind gift of the Jurkat cell line, Dr. Devi Vembu for the kind gift of cadaveric bone marrow, and Mr. Thomas Davis for the kind gift of K562, KGl, and KGla cells.
REFERENCES 1. Thomas M. V. (1982) Techniques 138, Academic Press, London/New
in Calcium York.
Research,
pp. 59-
MILLAR
AND
2. Campbell, 3. 4. 5. 6. 7.
A. K., Daw, R. A., Hallett, M. B., and Luzio, J. P. (1981) Biochem.J. 194,551-560. Ashley, C. C., and Campbell, A. K. (Eds.) (1979) Detection and Measurement of Free Calcium in Cells, Elsevier/North-Holland Biomedical Press, Amsterdam. Tsien, R. Y. (1980) Biochemistry 19,2396-2404. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260,3440-3450. Simpkins, C. O., Alailima, S. T., Tate, E. A., and Johnson, J. (1986)J. Surg.Res. 4 1,645-652. Tsien, R. Y., Pozzan, T., and Rink, T. J. (1982) J. Cell Biol. 94,
325-334. 8. Martell, A. E., and Smith,
R. M. (1974) Critical Stability Constants, Vol. 1, Plenum, New York. 9. Hallett, M. B., and Campbell, A. K. (1984) Cell Calcium 5,1-19. D. W., Chang, F.-H., Gifford, L. A., Goetzl, E. J., and 10. Goldman, Bourne, H. R. (1985) J. Exp. Med. 162,145-156. J. E., and Sheterline, P. (1985) Biochem. J. 231, 62311. Rickard,
628. 12. Andersson, T., Dahlgren, C., Pozzan, T., Stendahl, O., and Lew, P. D. (1986) Mol. Pharmcccol. 30,437-443. 13. Di Virgilio, F., Lew, P. D., Andersson, T., and Pozzan, T. (1987) J. Biol. Chem. 262,4574-4579. 14. Perez, H. D., Marder, S., Elfman, F., and Ives, H. E. (1987) Bio&em. Biophys. Res. Commun. 145.976-981.
ANALYTICALBIOCHEMISTRY
Quin-2 and FuraD. L. Mazorow2
(1990)
Measure Calcium Differently’
and D. B. Millar*
Immunobiology and Transplantation Department, Naval Medical Research Institute, Bethesda, Maryland 20814, and *Laboratory of Molecular Genetics, National Institutes of Mental Health, St. Elizabeth’s Hospital, Washington, DC 20032
Received
July
24,1989
Several fluorescent probes have been used in the past to monitor and to measure intracellular calcium and calcium fluxes. The most widely used of these probes are those developed by Tsien. We address the markedly different values obtained when comparing Quin-2 (the original probe) with Fura(a second-generation probe). In most cases the values for intracellular calcium have been considered to be interchangeable for the different probes. Using several different hematopoietic cell lines we show that in no case do the two o 1990 Academic PRSS, IIIC. probes yield equivalent values.
The detection and measurement of intracellular calcium is of the upmost importance to understanding cell function. To date the most consistent information has been obtained from a variety of fluorescent probes. For years the arsenazo dyes (1) as well as the photoproteins obelin (2) and aequorin (3) have been used to monitor changes in intracellular calcium. The disadvantage of these probes is that the cell needs to be permeabilized to allow entry of the probe. However, in 1980 Tsien (4) described a new fluorescent probe used to monitor intracellular calcium events. This new probe was called Quin2. The beauty of this technique was that the probe was available as a cell-permeable ester (Quin-2 AM). Once inside the cell, cytosolic esterases would deesterify the Quin-2 AM into Quin-2 which is not readily membrane permeable. Thus, the probe was placed in the cytosol without undue trauma to the cell membrane. However, Quin-2
has
a relatively
high
affinity
for
calcium
which
has generated concern that Quin-2 may strip calcium 1 The opinions here are the sole responsibility of the authors and in no way constitute official U.S. Navy policy. This work was supported by Navy Research and Development Command Work Unit MRO 4120-06002. * Present address: Environmental Medicine Department, Naval Medical Research Institute, Bethesda, MD 20814. To whom correspondence should be addressed. 28
from cell stores, yielding misleadingly high values for cytosolic calcium. Additionally Quin-2 bleaches appreciably over time which increases the difficulty in obtaining accurate values for F,,,,, and Fmi,. The advantage of Quin-2 over the other fluorescent probes mentioned is the ease of entry into the cell relative to the other calcium-sensitive probes described above. A few years later Tsien’s lab developed several other calcium-sensitive fluorescent probes which had improved capabilities compared to the Quin-2 probe (5). These were called Indo-l and Fura-2. These probes had a higher quantum yield than Quin-2, did not bleach as readily, and were more easily calibrated to “actual” calcium concentrations. Also, these probes had a more specific affinity and a lower binding constant than Quin-2 for calcium. We present here a comparison of Quin-2 and Fura- measurements of cytosolic calcium which suggest that the concerns over Quin-2 may be justified. METHODS
AND
MATERIALS
Neutrophils were isolated by standard procedures (6). Tissue culture cells (Jurkat, a human T-cell lymphoma cell line, K562, KGl, and KGla human myeloid leukemia cell lines) were grown in RPM1 1640 (Hazelton), 10% fetal calf serum (Hazelton), 2 mM glutamine (Cellgro), and penicillin (50 III/ml)-streptomycin (50 pg/ml) (Cellgro). The cells were incubated with either Quin-2 AM (Calbiochem) or Fura- AM (Calbiochem) for 30 min in a CO, incubator (37°C). Cells were centrifuged and resuspended to a final concentration of 5 X lo5 cells/ ml in Hanks’ balanced salt solution (without phenol red) with 15 mM Hepes.3 Resting calcium levels with Quin-2 were determined by the technique described by Grynkiewicz et al. (5). Briefly, [Cal = Kd(F - Fmi,)/(Fmax - F). The dissociation constant Kd = 115 nM, F is the measured fluorescence, and Fmin and F,,, are the minimum 3 Abbreviations used: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, EGTA, ethylene glycol bis(j3-aminoethyl N,N’-tetraacetic acid. 0003-2697/90
Copyright All rights
0 1990 by Academic
of reproduction
in any form
ether)$3.00
Press, Inc. reserved.
CALCIUM
MEASUREMENT
WITH
QUIN-2
The dissociation constant Kd = 135 nM, R is the measured ratio, Rmin and R,,, are the minimum and the maximum ratios obtained using Fura(acid form) in salts without calcium (10 mM EGTA) and with calcium (1 mM), Sfl is the measurement of free dye at the second excitation wavelength (in low-calcium salts), and Sb2 is the measurement of bound dye (in high-calcium salts) at the second excitation wavelength. The second technique is a direct calibration of fluorescence intensity using solutions of known calcium concentration made from mixtures of calcium salt and EGTA according to the stability constants of Martell and Smith (8) at a known pH, temperature, and ionic strength (5). The calibration also has an adjustment for pH, temperature (using the Van% Hoff equation), and ionic strength (values published by Tsien). The two methods of determining calcium concentration with Furaagreed within error, and our values agreed with those published by Grynkiewicz et al. (5) within 5 nM. Each calcium concentration was determined from at least five separate samples and the standard deviations and error were determined. Fluorescence measurements were obtained in a Perkin-Elmer Model 44B spectrophotofluorometer equipped with stirring and temperature control. All values obtained were at 37°C unless otherwise indicated. Quin-2 measurements were made with an excitation wavelength of 339 nm (slit 4 nm) and emission was determined at 492 nm (slit 6 nm). The Furastudies used excitation wavelengths of 335 and 362 nm (slit 4 nm) and emission was monitored at 510 nm (slit 6 nm). To check the accuracy of the system relative to the published values of Tsien, Furameasurements were also obtained at excitation wavelengths of 340 and 380 nm with emission set at 510 nm. Our values were in excellent agreement with those of Grynkiewicz et al. (5). RESULTS
In no case did measurements of Quin-2 and Furagive equal results. The most dissimilar of the results is the variance between Quin-2 and Furain neutrophils. The literature on neutrophil intracellular calcium levels using Quin-2 abound with values varying from 100 up to 200 nM (9-13). Our calcium values for neutrophils using
29
FURA-P TABLE
and maximum fluorescence, respectively. F,,, is obtained by sonically lysing the cells and releasing the intracellular Quin-2. Fmin is obtained by adding EGTA (10 mM final) (Fluka puriss) to the sonically disrupted solution and adjusting fluorescence intensity for change in volume. Calcium concentrations with Furawere determined using two somewhat independent means. The first is described by Tsien et al. (7) and consists of the equation
[Cal = & [(R - Rrnin)I(Rmax - ~)1(&2/&~2)-
AND
1
Comparison of Cellular Resting Calcium between Quin-2 and FuraCa concentration
(nM)
FuraCell type Neutrophil K562 Cadaveric Jurkats KG1 KGla
Quin-2
bone marrow
’ Intracellular was determined sity and solutions tures of calcium * Intracellular by Grynkiewicz
160 63f 72 102 95 73
+ 70 5 k 20 f 14 + 30 + 25
Standard
curve”
16f 3 50 f 10 36t 4 60f 5 32f 3 29+ 2
Equationb 14 55 43 54 37 35
f t i k + +
3 8 4 5 6 7
calcium was determined using a standard curve which using a direct calibration of Furafluorescence intenof known calcium concentration made from mixsalt and EGTA. calcium was determined using the equation described et al. (5) with R, &in, R,,,, Sn/Sb:!, and Kd = 135 nM.
Quin-2 were between 90 and 230 nM. However, we are not the only ones who obtained a very low value for resting calcium in neutrophils using Fura-2. Perez et al. (14) found resting values of calcium at 14 nM. Using either technique of Fura- analysis, the values we obtained for resting calcium were quite low. The resting calcium was between 11 and 19 nM. Our values were obtained from 12 different individuals. We also tested several other hematopoietic cells and cell lines (see Table 1). In Table 1 we compare the resting calcium levels of Quin-2 and Fura-2. In every case Quin-2 fluorescence yielded values which indicated higher cytosolic calcium levels than Fura-2. In all cases except K562 cells, the levels of cytosolic calcium using Quin-2 were at least twice the levels of Fura-2. DISCUSSION Although for many studies it is not essential to know the absolute calcium concentration within a cell, but to monitor changes in the cytosol after treatment with a ligand, it is important to note that equivalent values are not obtained with the different calcium probes, We wish to show not only that the indicated resting calcium levels of various hematopoietic cells differ among each other, but also that dissimilar values are obtained when using Quin-2 or Fura-2. There are many possible reasons for this. To measure Quin-2 one assumes that the pH and ionic strength within and without the cell are the same (which is not true). The other fact that needs to be remembered is that Quin-2’s affinity for calcium is within the same magnitude as that of many biological functions for calcium. Therefore, Quin-2 may shift the equilibrium of the cytosol as well as strip cytosolic stores of bound
30
MAZOROW
calcium. In the case of the neutrophil, Quin-2 may be pulling calcium from the granules as well as from other places. For the primitive relatively undifferentiated K562 cell line, the agreement between Quin-2 and Fura2 may reflect less complex modes of calcium storage. In conclusion, if one is only interested in a relative increase or decrease in cytosolic calcium, either of the probes should prove to be adequate, although Fura- will be more sensitive to small changes. However, if one is trying to establish true “values” for cytosolic calcium, then Fura- is a better choice. Also, Fura- is less toxic to the cell and may give a picture of events which more closely resembles true conditions. ACKNOWLEDGMENTS We thank Dr. Carl June for the kind gift of the Jurkat cell line, Dr. Devi Vembu for the kind gift of cadaveric bone marrow, and Mr. Thomas Davis for the kind gift of K562, KGl, and KGla cells.
REFERENCES 1. Thomas M. V. (1982) Techniques 138, Academic Press, London/New
in Calcium York.
Research,
pp. 59-
MILLAR
AND
2. Campbell, 3. 4. 5. 6. 7.
A. K., Daw, R. A., Hallett, M. B., and Luzio, J. P. (1981) Biochem.J. 194,551-560. Ashley, C. C., and Campbell, A. K. (Eds.) (1979) Detection and Measurement of Free Calcium in Cells, Elsevier/North-Holland Biomedical Press, Amsterdam. Tsien, R. Y. (1980) Biochemistry 19,2396-2404. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260,3440-3450. Simpkins, C. O., Alailima, S. T., Tate, E. A., and Johnson, J. (1986)J. Surg.Res. 4 1,645-652. Tsien, R. Y., Pozzan, T., and Rink, T. J. (1982) J. Cell Biol. 94,
325-334. 8. Martell, A. E., and Smith,
R. M. (1974) Critical Stability Constants, Vol. 1, Plenum, New York. 9. Hallett, M. B., and Campbell, A. K. (1984) Cell Calcium 5,1-19. D. W., Chang, F.-H., Gifford, L. A., Goetzl, E. J., and 10. Goldman, Bourne, H. R. (1985) J. Exp. Med. 162,145-156. J. E., and Sheterline, P. (1985) Biochem. J. 231, 62311. Rickard,
628. 12. Andersson, T., Dahlgren, C., Pozzan, T., Stendahl, O., and Lew, P. D. (1986) Mol. Pharmcccol. 30,437-443. 13. Di Virgilio, F., Lew, P. D., Andersson, T., and Pozzan, T. (1987) J. Biol. Chem. 262,4574-4579. 14. Perez, H. D., Marder, S., Elfman, F., and Ives, H. E. (1987) Bio&em. Biophys. Res. Commun. 145.976-981.