Calcium Signaling Protocols

343 Pages·1999·5.126 MB·English

by  Alec W. M. Simpson (auth.), David G. Lambert (eds.)

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Fluorescent Measurement of [Ca2+] 3
c
1
Fluorescent Measurement of [Ca2+]
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Basic Practical Considerations
Alec W. M. Simpson
1. Introduction
It is extremely difficult to write a prescriptive account of how to measure
cytosolic-free Ca2+([Ca2+] ) that will suit all potential investigators, given the
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wide diversity of fluorescent Ca2+indicators that are now available, the variety
of cells to be investigated, and an increasing range of detection equipment that
can be used. Therefore, this chapter is designed to provide the user with suffi-
cient background in the technology so that he or she can move toward develop-
ing a protocol that will suit the cells, the experimental objectives, and the
equipment available to the investigator.
The main approaches to measuring [Ca2+] before the synthesis of fluores-
c
cent Ca2+ indicators involved using the Ca2+-activated photoprotein aequorin,
Ca2+-selective microelectrodes, or absorbance indicators (1). The use of
aequorin and microelectrodes was generally restricted to large cells (usually
from invertebrates) that were easy to handle and manipulate with micropipets.
With a few notable exceptions (e.g., injection of hepatocytes and myocytes
with aequorin by Cobbold and colleagues [2,3]), these approaches were not
applied to the wide diversity of cells present in mammalian tissues. The use of
absorbance dyes did not become widespread since they are not very sensitive
to the typical [Ca2+] found in cells, and did not offer any real potential for
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investigating [Ca2+] in monolayers or single cells.
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The synthesis of quin2 by Tsien (4,5) in the early 1980s heralded a new
era in the measurement of Ca2+, by making available fluorescent probes that
could be readily introduced into living cells. The most commonly used
fluorescent Ca2+ indicator at present is fura-2, which, along with indo-1,
From:Methods in Molecular Biology, Vol. 114: Calcium Signaling Protocols
Edited by: D. G. Lambert © Humana Press Inc., Totowa, NJ
3
4 Simpson
formed a new generation of ratiometric indicators also designed by Tsien and
colleagues (6).
The Ca2+-binding properties of these indicators is formed by the presence of
a tetracarboxylic acid core as found in the Ca2+-chelator EGTA. Whereas the
Ca2+ binding of EGTA is highly pH dependent, the original Ca2+ indicator
quin2 and its successors were designed around an EGTA derivative, BAPTA,
also synthesized by Tsien (7). For a compound to act as an intracellular Ca2+
indicator, selectivity of the indicator for Ca2+ over other physiologically
important ions is essential. EGTA already showed a much greater selectivity
for Ca2+over Mg2+, Na+and K+, but unfortunately, its Ca2+binding is very pH
sensitive. Cells undergo physiological changes in pH (8), which in the case of
an EGTA-like chelator would affect the reported [Ca2+]. Calibrating a pH-sen-
sitive Ca2+indicator would be quite difficult, since small changes in pH of the
calibration solutions would affect the measured fluorescence and the K for
d
Ca2+. The synthesis of BAPTA, a largely pH-insensitive Ca2+ chelator, was
therefore an important step in the development of fluorescent probes for mea-
suring [Ca2+] (7).
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Since the introduction of quin2, fura-2, and indo-1, numerous other fluores-
cent Ca2+ indicators have been synthesized, each with varying fluorescence
characteristics and K s for Ca2+seeref.9;Tables 1–3). The fundamental prop-
d
erties of these indicators are similar, in that the binding of Ca2+ produces a
wavelength shift in either the excitation or emission fluorescence spectra (6,9).
When there is little or no shift in the excitation spectra, a Ca2+-dependent
change in the emission intensity is used to report changes in Ca2+ (5,9). This
can arise from Ca2+-dependent changes in the intensity of absorbance or quan-
tum efficiency.
In terms of fluorescence properties, the indicators can be divided into two
main groups, those that are excited by near ultraviolet (UV) wavelengths
340–380 nm (e.g., quin2, fura-2, and indo-1) and those that are excited with
visible light at or above 450 nm (e.g., fluo-3, Calcium Green, rhod-2; seerefs.
9 and 10) The fluorophores for the visible indicators tend to be fluoroscein
and rhodamine derivatives. This is advantageous since a great deal of fluo-
rescence instrumentation has been designed for use with fluoroscein- and
rhodamine-based dyes.
2. Overview
2.1. Single Excitation Indicators
The first of this family is quin2, Tsien’s (5)original fluorescent Ca2+indica-
tor. When excited at 340 nm, an increase in emission intensity peaking at 505 nm
is observed on binding Ca2+. Under physiological conditions, quin2 has a K of
d
Fluorescent Measurement of [Ca2+] 5
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115 nM, making it useful for measuring [Ca2+] changes at or close to those
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found in unstimulated (resting) cells; however, the dye is of little use in moni-
toring changes in [Ca2+] in excess of 1 µM. Poor quantum efficiency has
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limited the use of this indicator, especially after the introduction of the more
fluorescent ratiometric probes. However, quin2 does have some useful proper-
ties; like BAPTA, it is a very good buffer of [Ca2+] , and its use has allowed
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Ca2+-independent phenomena to be observed (11,12). Subsequently improved
single excitation indicators have been developed that are more fluorescent
and have K s for Ca2+ between ~200 nM and 20 µM (9,10,13) (see Table 1).
d
These indicators include fluo-3 (10), and the Calcium Green and Calcium Orange
series of indicators. With these indicators there is little or no shift in either
the excitation or emission spectra; however, a marked increase in fluorescence
intensity can be observed on Ca2+ binding. Calcium Green-2™ has a K of
d
550 nM (Table 1) and produces approx 100-fold increase in fluorescence
between being Ca2+-freeand Ca2+-saturated. For fluo-3 this increase is reported
to be approx 200-fold. The fluo and Calcium Green indicators all have peak
excitation spectra at or close to 490 nm (see Table 1), allowing them to be
readily used with argon-ion lasers (488 nm excitation). Peak emission lies
close to 530 nm. There are Ca2+indicators that can be excited even at longer
wavelengths, e.g., rhod-2, the Calcium Crimson and Calcium Orange series,
and KJM-1 (Table 1). Rhod-2 is excited at 520 nm, with a peak emission at
580 nm (10), and has been used to measure mitochondrial Ca2+ rather than
[Ca2+] (14). Fura-Red (strictly a ratiometric indicator) when excited at wave-
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lengths close to 480 nm can be used in combination with fluo-3 to obtain a
ratio derived from their respective 530- and 650-nm emission signals. Thus,
combinations of visible excitation indicators can be used to obtain ratio mea-
sures of [Ca2+] (15,16).
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2.1.1. Visible Excitation Indicators
Visible wavelength indicators are attractive because they can avoid problems
such as light absorbance by optical elements and cellular autofluorescence.
The lower excitation energies of the longer wavelengths also means that
photobleaching is reduced. The visibly excited dyes are more suited to the laser-
based illumination systems used in confocal microscopy and flow cytometry.
The advantage of having a range of indicators that can be excited at different
wavelengths is that combinations of ion-indicators can be used together. Thus,
Ca2+ can be monitored simultaneously with other physiologically important
ions such as Na+ or H+(17–19). Moreover, Ca2+ can be monitored using indi-
cators in separate domains as with simultaneous measurements of intracellular
and extracellular Ca2+ (20).
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Table 1
Single Excitation Wavelength Indicators
Absorbance Emission
AM
Indicator Sourcea UV/V K loading –Ca2+ +Ca2+ –Ca2+ +Ca2+ Comments
d
Quin2 MP/TL UV 60b(115)d ✔ 352 332 492 498 High intracellular buffering
Methoxyquin2MF MP UV 65b ✔ 352 332 492 498 MethoxyquinMF19 for nuclear
magnetic resonance
Oregon Green 488 MP V 170b ✔ 494 494 523 523 Designed for argon-ion lasers
BAPTA-1
Calcium Orange™ MP V 185b(380)e ✔ 549 549 575 576 Seeref.13
Calcium Crimson™ MP V 185b(221)c ✔ 590 589 615 615 Seeref.13
Calcium Green-1™ MP V 190b(221)e ✔ 503 506 534 533 Seeref.13; fluorescence lifetime
measurements
Calcium Green C MP V 280b(62)f ✘ 509 509 530 530 Near membrane Ca2+ indicator;
18
K affected by lipids; ref.53
d
Fluo-3 MP/TL V 390b ✔ 506 506 526 526 AM ester and Ca2+-free forms
only weakly fluorescent; large
increase in fluorescence on
Ca2+ binding
KJM-1 TL V 500c ✔ 560 560 Em640
Calcium Green-2™ MP V 550 ✔ 503 503 536 536
Fluo LR TL V 550c ✔ 506 506 525 525 Leakage-resistant indicator
Rhod-2 MP/TL V 570b ✔ 549 552 571 571 Can be loaded as dihydro
derivative; will locate in
S
mitochondria and peroxisomimes
Oregon Green 488 MP V 580b ✔ 494 494 523 523 Designed for argon-ion lasersp
s
BAPTA-2 o
n
Fluo-535 TL V 800 ✔ 535 535 560 560 Lower-affinity derivative F
lu
(Fluo-535FF) also availableo
Magnesium Green™ MP V 6 µMb ✔ 506 506 531 531 K in 0-Mg2+; indicator will bree
d s
Mg2+ sensitive c
e
Calcium Green-5N™ MP V 14 µMb ✔ 506 506 532 532 n
t
Calcium Orange-5N™ MP V 20 µMb ✔ 549 549 582 582 Exhibits very fast kinetics M
suitable for millisecond timee
a
resolution; ref.77 s
u
Oregon Green 488 MP V 20 µMb ✔ 494 494 521 521 Designed for argon-ion lasersre
m
BAPTA-5N™
e
Fluo-3FF TL V 41 µMb ✔ Ex 515 526 526 n
t

o
aMP, Molecular Probes; TL, Teflabs; MP/TL, Molecular Probes, Teflabs, and other suppliers. f

bK determined in 100 mM KCl, pH 7.2, at 22°C. [C
d
cK determined at pH 7.2 and 22°C. a
d 2
dK determined in 100 mM KCl, pH 7.05, at 37°C. ]+
d c
eValues taken from ref.13.
fK reported to be 230 nM at 0.1M ionic strength, pH 7.2, at 22°C and 62 nM in the presence of phospholipid vesicles. Seeref.50.
d
7
Table 2 8
Dual Excitation Ratiometric Indicators
Absorbance Emission
AM
Indicator Sourcea UV/V K loading –Ca2+ +Ca2+ –Ca2+ +Ca2+ Comments
d
Fura Red™ MP V 140b ✔ 472 436 657 637 Low quantum yield; used in
combination with single ex
indicators to obtain ratios
Fura-2 MP/TL UV 145b(224)c ✔ 363 335 512 505
Fura C MP UV 150b ✘ 365 338 501 494 Lipophilic near-membrane
18
Ca2+ indicator
Fura PE3 TL UV 250d ✔ 364 335 508 500 Leakage-resistant indicator
FFP18 TL UV 400 ✔ 364 335 502 495 Lipophilic near-membrane
Ca2+ indicator
8 Bis-fura-2 MP UV 370b(525)e ✘ 366 338 511 505 Brighter and has lower affinity
than fura-2; not available as
an ester
BTC MP V 7 µMb ✔ 464 401 533 529 Visible-excitation ratiometric
indicator
Mag-fura-2 MP UV 25 µMb ✔ 369 329 511 508 K in 0-Mg2+; indicator will be
d
(Furaptra) Mg2+ sensitive
Mag-fura-5 MP UV 28 µMb ✔ 369 330 505 500 K in 0-Mg2+; indicator will be
d
Mg2+ sensitive
Fura-2FF TL UV 35 µMd ✔ 335 364 512 505
aMP, Molecular Probes; TL, Teflabs; MP/TL, Molecular Probes, Teflabs, and other suppliers.
bK determined in 100 mM KCl, pH 7.2, at 22°C. S
cKd determined in 100 mM KCl, pH 7.05, at 37°C. im
d p
dConditions for K determined not defined. s
d o
eConditions same as in footnote b, but with 1 mM Mg2+ present. n
F
lu
o
r
e
s
c
e
n
t

M
Table 3 e
a
Dual Emission Ratiometric Indicators s
u
r
Absorbance Emission e
AM m
e
Indicator Sourcea UV/V Kd loading –Ca2+ +Ca2+ –Ca2+ +Ca2+ Comments n
t

Indo-1 MP/TL UV 230b(250)c ✔ 346 330 475 401 of

Indo PE3 TL UV 260d ✔ 346 330 475 408 Leakage-resistant indicator [C
FIP18 TL UV 450d ✔ 346 330 475 408 Lipophilic near-membrane a2
+
9 Ca2+ indicator c]
Indo-1FF TL UV 33 µMd ✔ Ex 348 475 408
Mag-indo-1 MP UV 35 µMe ✔ 349 328 480 390 K in 0-Mg2+;indicator will be
d
Mg2+ sensitive
aMP, Molecular Probes; TL, Teflabs; MP/TL, Molecular Probes, Teflabs, and other suppliers.
bK determined in 100 mM KCl, pH 7.2, at 22°C.
d
cK determined in 115 mM KCl, 20 mM NaCl, 10 mM K-MOPS, pH 7.05, 1 mM Mg2+ at 37°C.
d
dConditions for K determination not defined.
d
eK determined in 100 mM KCl, 40 mM HEPES, pH 7.0, at 22°C.
d
9
10 Simpson
Fig. 1. Ca2+-free and Ca2+-saturated excitation spectra of fura-2. The two spectra
coincide at 360 nm, the isobestic (or isoemissive) point. It can be seen that when Ca2+
binds, the fluorescence signal will increase when the indicator is excited at 340 nm,
remain the same when it is excited at 360 nm, and decrease when it is excited at 380 nm.
2.1.2. Caged Compounds
Bioactive molecules can be incorporated into physiologically inert (caged)
molecules and subsequently released in a controlled manner by photolysis of
the chemical “cage.” Introduction of the visible excitation indicators has
allowed [Ca2+] to be measured during UV-induced flash photolysis of caged
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compounds such as caged Ins(1,4,5)P and Nitr-5 (caged Ca2+ ) (21). This
3
advance has enabled second messengers to be manipulated in a controlled man-
ner while simultaneously monitoring [Ca2+] .
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2.2. Dual Excitation Indicators
Fura-2 is the archetypal dual excitation Ca2+indicator(6). In low Ca2+, fura-2
shows a broad excitation spectrum between 300 and 400 nm, with a peak at
approx 370 nm. On Ca2+binding, the excitation peak increases in intensity and
also shifts further into the UV (Fig. 1). Consequently, if the dye is excited at
340 nm (emission monitored at 510 nm), Ca2+binding will produce an increase
in fluorescence, whereas a decrease in the fluorescent signal is observed when
the dye is excited at 380 nm (Figs. 1 and 2). When the dye is excited in quick
Fluorescent Measurement of [Ca2+] 11
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Fig. 2. The typical signals obtained from a fura-2–loaded cell when it is excited
alternately at 340 and 380 nm. Agonist stimulation will cause an increase in the 340-nm
signal and a decrease in the 380-nm signal. Addition of a Ca2+ionophore (Iono), in the
presence of Ca2+, will give F andF , whereas subsequent addition of EGTA
340max 380min
will give F andF . The time taken to reach F andF after addition
380max 340min 340max 380min
of ionophore and EGTA can vary and be in excess of 30 min. Curve-fitting the decay
towardR has been suggested as a strategy to speed up the calibration process (34).
min
The long time period required to obtain R (340min/380max) is not ideal for imaging
min
experiments since the dimensions of the cell may change during the calibration.
succession at 340 and 380 nm, a ratio of the respective emission signals can be
used to monitor [Ca2+]. Ratiometric measurements have a number of advan-
tages over single wavelength probes. The ratio signal is not dependent on dye
concentration, illumination intensity, or optical path length. Therefore, spatial
variations in these parameters will not affect the estimations of [Ca2+] . Such
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factors are especially important if the dyes are to be used for imaging of [Ca2+] ,
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which illumination intensity and optical properties vary across the field of view
(6,22). Dye leakage and photobleaching frequently lead to a loss of indicator
during an experiment; thus, the active indicator concentration cannot be
assumed to be constant (23,24). Under such conditions, a ratiometric indicator
gives a more stable measure of [Ca2+] than could be obtained from a single
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excitation indicator. Ratiometric measurements also produce an additional
increase in sensitivity.
A further useful property of ratiometric indicators is the presence of an
isobestic or isoemissive point. For example, when fura-2 is excited at 360 nm,