Membrane 1

B I O I M A G I N G A P P L I C AT I O N N O T E
BD BiosciencesB I O I M A G I N G A P P L I C A T I O N N O T E
Simultaneous Measurement of [Ca2+]c And Mitochondrial

Membrane Potential in Astrocytoma Cells Using Two
Ratiometric Fluorescence Probes
Introduction Measurement of cytosolic, free Ca2+ concentration

Fluorescence measurements of intracellular events have [Ca2+]c has been employed successfully to unravel
become a staple for manual and automated intracellular signaling pathways, identify G-protein
microscopy. Cellular events have been measured either coupled receptors (GPCR) and, recently, to measure
by following an increase in fluorescence within a sub- cAMP levels by using specifically modified ion
cellular compartment (“intensity-based”) or by channels. Most assays are performed using single
observing the motion of a fluorescently-tagged protein wavelength dyes such as Fluo-3 and Fluo-4 which are
from one compartment into another (“translocation-
based”). These methods can be combined and,
depending on the assay, performed in live or fixed cells.
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Bioimaging Application Note - Calcium/Astrocytoma
easy to use, have large quantum yield, and are appropriate for both laser-
based and lamp-based systems (Molecular Probes, Handbook Section 20.3).
The most significant downside with using these dyes
is that the intensity signal generated is directly proportional to the number of
dye molecules loaded into the cell, a factor that is dye, concentration, cell-
type, and time-dependent and hence is prone to substantial day-to-day
variation. Ratiometric dyes like Fura-2 or Indo-1 overcome most of these
problems by exploiting the wavelength shift in the emission (Indo-1) or
excitation (Fura-2) upon binding of Ca2+. This shift is independent of the dye
concentration and can be extracted by taking two images in rapid succession
and dividing them to generate a ratio measurement. Unlike Fluo-3 and -4,
Fura-2 does not preferentially load into the nucleus and is therefore much
more suitable for estimating cytoplasmic fluorescence intensity (Thomas et al.
2000). A further advantage of Fura-2 is its UV excitation spectrum, leaving
the “green” channel (ordinarily used by e.g. Fluo-3 and 4 but also
by Green Fluorescent Protein and FITC) free for other dyes in multiplexing
experiments.
One of the earliest signs of impending apoptosis is the loss of mitochondrial

membrane potential (Emm,; Zuliani et al., 2003). This early marker has been
exploited in both flow-cytometry as well as microscopy-based assays as a
general marker of cell health, especially in toxicology assays. The fluorescent
probe JC-1 specifically loads into the mitochondrial membrane and forms
red-fluorescent aggregates (“J-aggregates”) when the membrane potential is
high. Loss of membrane potential is associated with a loss of aggregation,
and the reduction in the red emission intensity can be observed along with a
signal increase in the green spectrum. This is due to the monomeric form of
the dye which mainly localizes in the cytosol. Aggregation of the probe is also
concentration-dependent and measurements of JC-1 fluorescence changes are
therefore preferably ratiometric.
Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale.
2 bdbiosciences.com All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.
The interactions between mitochondrial function and Ca2+ homeostasis are very
complex (see Ganitkevich, 2003 for review on mitochondrial [Ca2+]c and Berridge et
al. 1998 for review on [Ca2+]c in apoptosis) and are the subject of ongoing research.
Apoptosis-inducing treatments such as photodynamic therapy (PDT) can directly
influence [Ca2+]c homeostasis (Granville et al. 2001) and mitochondrial function.
BD Biosciences has developed the BD Pathway™ Bioimager, a sophisticated automated
cellular imaging platform for advanced academic research and assay development. The
BD Pathway Bioimager uses lamp excitation and can take advantage of the entire
visible, near-UV, and near-infrared spectrum for excitation of multiple probes. The
system’s proprietary confocal disk can be switched into and out of the light path by
the user in order to accommodate virtually all imaging needs.
The BD Pathway Bioimager is very suitable for the study of living cells due to its
environmentally controlled chamber and its sophisticated liquid handling system.
Drugs can be dispensed from a drug treatment plate by using disposable tips,
obviating the need for a distinct needle wash station. The BD Pathway Bioimager
system allows drugs to be dispensed while cells are imaged, thus allowing the
continuous observation of rapid cellular events such as changes in [Ca2+]c or Emm.
The goal of these experiments was to demonstrate how the BD Pathway Bioimager
system can be used in the study of [Ca2+]c and Emm. We specifically chose two
ratiometric dyes, Fura-2 (dual excitation dye) and JC-1 (dual emission dye) in order
to eliminate dye loading artifacts and to demonstrate that the BD Pathway Bioimager
system is designed to allow multiplexing on a cellular level even under complex
imaging conditions.
Methods
Cell Culture
Rat Glioma Cells (C6-2B clone, Munshi et al. 1993) were grown and maintained in
Minimum Essential Medium, Eagle’s, Dulbecco’s Modification (DMEM, Biofluids,
P104G) containing 10% Fetal Bovine Serum (Gibco, 26140-079), 1% Penicillin /
Streptomycin (Biofluids, 303). For experimentation, 7000 cells/well were seeded into
96 well plates (Costar #, 3614) and grown overnight at 37º C, 5% CO2 / 95% Air.
Dye Loading
Cells were co-loaded with 5 µM Fura-2/AM (Molecular Probes, F1201) and 5 µg/mL
JC-1 (Molecular Probes, T3168) in growth medium for 30 min at 37º C. Cells were
then washed twice with phenol-red free HBSS (Hanks Balanced Salt Solution,
containing 10 mM HEPES, pH 7.2) to remove excess dye and were then imaged.
Note: These experiments were performed without the use of a nuclear dye.
Imaging
Plates were moved into the environmental chamber of the BD Pathway 855 Bioimager.
The drug treatment plate was placed into its holder where it is accessible to the
3-dimensional automated dispenser. The dispenser uses disposable tips, obviating the
need for a wash station. This allows for the addition of the drug directly above the
imaging station and during a time course, without having to move the sample plate.
Drugs were added as a bolus in non-contact mode. Although the system is capable of
active mixing, this step was found unnecessary in this experiment (data not shown).
Cells were imaged at 37ºC and under 5%CO2/95% Air. The cells were imaged using
an Olympus 20X Uapo/340, 0.75 na objective. This objective was chosen due its
superior transmission properties in the Fura-2 excitation channels.
All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details. bdbiosciences.com 3
The imaging protocols used the following filter wheel settings:

Table 1 Filter Settings for Fura-2 / JC-1 Ratiometric Imaging in the
BD Pathway Bioimager
Dye Excitation Dichroic Emission

Fura-2 340nm 400DCLP 435LP
dual excitation ratio 380nm
JC-1 488nm Fura FITC Fura-FITC

dual emission ratio 570LP
Pharmacological Treatment
Two sets of experiments were conducted. The first experiment investigated the effects
of carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP, 500 nM at t = 26s)
followed by the addition of ATP (100 µM at t = 125s). FCCP is a known
protonophore with pronounced effects on mitochondrial membrane potential (Emm).
Interrupting the proton transport across the inner mitochondrial membrane results in
a complete cessation of ATP production and hence the interruption of major cellular
events. Although FCCP is primarily used to study mitochondria signaling, Tretter et al.
(1997) have conclusively shown that under specific circumstances, it can also increases
[Na+]c which in turn affects [Ca2+]c.
In the second experiment, ATP was added at t = 26s and valinomycin (a K+
ionophore, 1 µM) at t = 125s.
Results
Dye Loading
In addition to the dye concentration achieved in living cells, the location of the dye
molecules is of great importance. Parameters such as temperature, the chemical nature
and concentration of the dye, as well as the cell type, all affect dye loading and have
to be carefully assessed to ensure that the measured signal is indeed indicative of the
specified organelle being investigated (Thomas et al. 2000).
The C6-2B cells in Figure 1 show very specific mitochondrial loading of the JC-1 dye.
This is an enlargement of the image shown in Figure 2 (left panel).
The BD Pathway Bioimager is the only system where users can switch between
confocal and widefield illumination where the assay demands it. Although confocality
superbly reduces the background fluorescence in cellular screening, the presented dyes
showed a very good signal/noise ratio and hence did not warrant the use of the
confocal disk.
Figure 1 JC-1 loaded mitochondria in C6-2B cells at 20X magnification

(area enlarged from Figure 2)
FCCP-induced Changes in [Ca2+]c and Mitochondrial Membrane Potential (Emm)

C6-2B cells treated with FCCP showed a pronounced increase in the JC-1 520/580nm
emission ratio intensity. FCCP is known to uncouple the mitochondria electron
transport chain and a loss of Emm is expected as a consequence. Figure 2 shows the
pseudo-color overlay of the FCCP response in JC-1 loaded cells. The upper panel
shows the cells as predominantly “red”, that is, the J-aggregates outnumber the single
dye form indicating intact Emm. Once FCCP is added and Emm collapses, J-aggregates
dissolve and the cells appear “green” (lower panel).
At the same time, the 340/380nm excitation ratio of Fura-2 changed only marginally
and the [Ca2+]c response is quite heterogeneous amongst the cells. The loss of Emm
does, therefore, not directly evoke a [Ca2+]c response, at least not when the signal is
expressed as the average of all cells in the field of view (Figure 3).
The average responses are shown in Figure 3. Here, the uncoupling of the
mitochondrial electron transport chain results in a shift of the JC-1 fluorescence (also
shown in Figure 2). This loss of Emm is accompanied by a small and variable [Ca2+]c
response. Even the addition of ATP at the end of the experiment could not evoke any
further [Ca2+]c response, suggesting that the cells were rendered unresponsive due to
the uncoupling of the mitochondrial electron transport chain or, alternatively, that the
Ca2+ stores could be empty.
Figure 2 FCCP induces a strong

depolarization of the
mitochondrial membrane
potential shown by the JC-1
ratio fluorescence intensity.
The images are color
overlays of JC-1 emission
spectra at 580nm (red)
and 520nm (green)
Figure 3 Ratiometric Assessments of Both [Ca2+]c and Emm in FCCP-challenged
C6-2B Cells: Shown are mean ± SEM with n= 72 cells.
A screenshot of the AttoViosion software interface shows how the system records and
presents data along with images (Figure 4). The top left panel (pseudo-colored blue)
shows the Fura-2 loaded cells, below is the corresponding green-colored JC-1 image.
Each cell is outlined by a “Region of Interest” (ROI) automatically generated by the
software and the fluorescence intensity values of these ROIs in each channel (Fura-2
and JC-1) are collected and presented as kinetic traces (right panels). Here, the
respective upper panels show the ratio-ed cellular responses whereas the lower panels
show the raw traces as they are generated by the ROIs in the individual nominator
and denominator channels respectively. The traces themselves are interactive: selecting
a trace identifies its cell and vice versa.
The image sets are stored as individual 16 bit .tif files and can be “played” as movies
inside AttoVision and in many other sophisticated imaging software programs.
Figure 4 FCCP-induced Changes in Fura-2 and JC-1 Fluorescence Intensities

The traces show a rather heterogeneous response to FCCP in the [Ca2+]c channel
and more consistent response in the Emm channel. The discrepancy may be due to the
cellular signaling properties. FCCP is known to uncouple the Emm, the loss of which
may trigger a Ca2+ release from the mitochondria into the cytoplasm. In some cells,
the sudden rise of [Ca2+]c may trigger a Ca2+ - induced Ca2+ release (CICR) from the
endoplasmic reticulum and also Ca2+ influx from the extracellular space (Roderick
et al. 2003).
ATP and Valinomycin induced changes in [Ca2+]c and Emm

A second experiment was performed to assess the effect of ATP on both the Fura-2
and the JC-1 signal. ATP is known to mobilize Ca2+ from internal stores in C6-2B cells
(Munshi et al., 1993) and Figure 5 shows the robust increase in [Ca2+]c, probably due
a combination of a rapid and transient Ca2+ release from intracellular stores followed
by a slower and more sustained Ca2+ influx phase. ATP did not seem to affect Emm, as
shown by the average response of all the cells (Figure 5) or by the individual traces
(Figure 6).
Valinomycin, a K+ ionophore caused a rapid and sustained increase in JC-1
(520/580nm) fluorescence emission ratio indicating a complete and irreversible
collapse of the Emm. Valinomycin however did not cause any significant change in
[Ca2+]c.
Figure 5 Ratiometric Assessments of both [Ca2+]c and Emm in ATP and

Valinomycin-challenged C6-2B Cells
Figure 6 JC-1 emission ratios of C6-2B Cells Treated with ATP (100µM)
and Valinomycin
Correlation Between [Ca2+]c and Emm

To test the hypothesis as to whether the variable rise in [Ca2+]c caused by FCCP is
correlated with the loss of Emm, on a cell-by-cell basis, a correlation analysis was
performed.
AttoVision allows the user to segment the experimental time course into distinct
“Treatment Zones”, (TZ, see Figure 7, roman numerals). Each TZ contains the kinetic
response data associated with this interval. In the presented experiment, the first TZ
(I) was associated with the basal activity of the cells and ranged from the start of the
experiment to the time of the first drug addition (FCCP). The second phase was
allocated to span the FCCP response (II), the third interval extended from after the
FCCP response was complete until the addition of ATP (III). The last phase covers the
time between the ATP addition and end of the experiment (IV).
Figure 7 Use of Treatment Zones (TZ) in the Analysis of Kinetic Responses

(roman numerals, error bars omitted for clarity, see Figure 3)
The four kinetic parameters (maximum response, minimum response, mean response,
rate of rise) for each TZ and for each dye were exported as a text file and analyzed in
Microsoft Excel™. Since all analysis steps are performed on a cell-by-cell basis, this
method can identify specific subpopulations of cells.
The full (linear) correlation table would span all four TZ, four kinetic parameters and
two dyes. Therefore, it would present itself as a matrix of 32x32 to cover all possible
correlation coefficients.
Table 2 shows this correlation table (abridged to only TZ2 for clarity). Clear
correlations can be seen when the “rate of rise” in the Fura-2 channel is compared to
the “maximum response” in the same channel. This suggests that in those cells which
respond to FCCP with a rise in [Ca2+]c, the steepness of the rise is correlated to the
maximum response (r2 = 0.848).
However, the analysis of [Ca2+]c (Fura-2) parameters compared to those of the Emm
(JC-1) show no significant correlation (shaded area in Table 2). This suggests that
FCCP-induced [Ca2+]c changes are not correlated with the loss of mitochondrial
membrane potential, probably because Ca2+ and Emm follow different signaling
pathways.
When the analysis was extended over all four TZ, no significant correlation was
detected (data not shown).
Fura 2 JC-1 JC-1

Fura 2 Fura 2 Fura 2 Rate of Maximum JC-1 JC-1 Rate of
Maximum Minimum Average Rise Minimum Average Rise
Fura 2 Maximum 1.000

Fura 2 Minimum 0.032 1.000
Fura 2 Average 0.757 0.234 1.000
Fura 2 Rate of Rise 0.848 0.008 0.555 1.000
JC-1 Maximum 0.092 0.177 0.236 0.056 1.000
JC-1 Minimum 0.130 0.061 0.140 0.079 0.365 1.000
JC-1 Average 0.107 0.210 0.265 0.068 0.848 0.640 1.000
JC-1 Rate of Rise 0.002 0.102 0.077 0.001 0.560 0.004 0.244 1.000
Table 2 Correlation Analysis (r2) of Fura-2 and JC-1 Responses to FCCP
Discussion
The experiments presented are representative of a wide variety of novel assays in drug
research, functional biology, and SAR-studies.
The hypothesis was that uncoupled mitochondria rapidly leak Ca2+ into the cytoplasm and force
a Ca2+-induced-Ca2+-release. Cell-by-cell analysis showed that there was in fact no measurable
correlation between the magnitude of the Ca2+ and the Emm response. This suggests that FCCP
evokes an increase in [Ca2+]c by different pathways than those that uncouple the mitochondria.
On a practical note, the loss of Emm is often used as the first indicator of imminent apoptosis
or necrosis. The need to measure toxicity of putative drugs at an early stage spawned the
development of multiplexed apoptosis fixed-cell assays (e.g. caspase 3/5 binding) that can
not be used with living cells.
In addition, the most commonly used assays focus on late events in the apoptotic process and
may be of limited value to researchers aiming to see subtle or early events.
We show that [Ca2+]c and mitochondrial membrane potential can be measured simultaneously
by highly quantitative, ratiometric means without interference between the two biological
responses.
Live-cell assays where candidate GPCR agonists are tested simultaneously for their effect on Ca2+
homeostasis and Emm would represent valuable, early stage cytotoxicity screening tools.
References
1 Berridge MJ, Bootman MD, Lipp P. Calcium—a life and death signal. Nature 1998 Oct 395:645-8
2 Ganitkevich VY. The role of mitochondria in cytoplasmic Ca2+ cycling. Exp Physiol 2003 Jan
88:91-7
3 Granville DJ, Ruehlmann DO, Choy JC, Cassidy BA, Hunt DW, van Breemen C, McManus BM Bcl-2
increases emptying of endoplasmic reticulum Ca2+ stores during photodynamic therapy-induced
apoptosis. Cell Calcium 2001 Nov 30:343-50
4 Munshi R, DeBernardi MA, Brooker G. P2U-purinergic receptors on C6-2B rat glioma cells:
modulation of cytosolic Ca2+ and cAMP levels by protein kinase C. Mol Pharmacol 1993 Dec 44:6
1185-91
5 Roderick HL, Berridge MJ, Bootman MD. Calcium-induced calcium release. Curr Biol 2003 May
13:R425
6 Thomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P. A comparison of fluorescent
Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell
Calcium 2000 Oct 28:213-23
7 Tretter L, Chinopoulos C, Adam-Vizi V Plasma membrane depolarization and disturbed Na+
homeostasis induced by the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon
in isolated nerve terminals. Mol Pharmacol 1998 Apr 53:734-41
8 Zuliani T, Duval R, Jayat C, Schnebert S, Andre P, Dumas M, Ratinaud MH. Sensitive and reliable
JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma
membrane integrity: interest for cell death investigations. Cytometry 2003 Aug 54A:2 100-8
©2005 Becton, Dickinson and Company. BD, the BD Logo, and BD Pathway Bioimager are all
trademarks of Becton, Dickinson and Company.
This application note is for education purposes only. The use of the procedures, products, or
methods outlined in this application note may be protected by intellectual property owned by
others. A license may be required to practice the procedures described, and procuring such
license is the sole responsibility of the researcher.
Notes
Notes
Notes
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B I O I M A G I N G A P P L I C AT I O N N O T E

Simultaneous Measurement of [Ca2+]c And Mitochondrial

Introduction Measurement of cytosolic, free Ca2+ concentration

One of the earliest signs of impending apoptosis is the loss of mitochondrial

The imaging protocols used the following filter wheel settings:

Dye Excitation Dichroic Emission

JC-1 488nm Fura FITC Fura-FITC

Figure 1 JC-1 loaded mitochondria in C6-2B cells at 20X magnification

FCCP-induced Changes in [Ca2+]c and Mitochondrial Membrane Potential (Emm)

Figure 2 FCCP induces a strong

Figure 4 FCCP-induced Changes in Fura-2 and JC-1 Fluorescence Intensities

ATP and Valinomycin induced changes in [Ca2+]c and Emm

Figure 5 Ratiometric Assessments of both [Ca2+]c and Emm in ATP and

Correlation Between [Ca2+]c and Emm

Figure 7 Use of Treatment Zones (TZ) in the Analysis of Kinetic Responses

Fura 2 JC-1 JC-1

Fura 2 Maximum 1.000

Table 2 Correlation Analysis (r2) of Fura-2 and JC-1 Responses to FCCP

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