93

Kinetics
John Ransom, PhD
Defnition and Overview
Kinetics, or more precisely, Cellular Response Kinetics, refers to the study of cellular
signal transduction events that have relatively transient natures, on the order of a
several minutes or less. Typically, once initiated, the response rises to a peak level
within seconds, and the peak level may be sustained before declining, or it may begin
to decline soon after the peak is attained and at a relatively rapid rate.
To study kinetic events with a uorescence-based instrument like a ow cytometer, it is
necessary to have a uorescent probe that faithfully reects the transient nature of the
signal transduction event being studied. An excellent example is the family of probes
including indo-1, uo-4 and fura-2, designed to measure intracellular calcium levels
(Ca
2+
i
) in viable cells.
1
These probes have afnity constants for Ca
2+
that are slightly
higher than the corresponding concentrations of Ca
2+
in resting cells, exhibit changes in
their uorescent properties when the probe goes from the Ca
2+
-free to the Ca
2+
bound
state, are non-toxic, and can be easily loaded into viable cells. Thus, the probes will
indicate both an increase and a decrease in free cytosolic Ca
2+
within the viable cell
with minimal error in reporting the temporal aspects of the transient changes in Ca
2+
i
.
In ow cytometry work, kinetic studies are most often typied by the Ca
2+
i
mobilization
responses initiated by a wide variety of receptors that couple to mobilization of a Ca
2+

store within the cell, or to inux pathways that allow Ca
2+
to diffuse in from outside
of the cell down a very large and inwardly directed Ca
2+

concentration gradient. As
a potentially toxic ion, free Ca
2+

would be lethal to a cell if it were allowed to exceed
normal intracellular concentrations of approximately 100 nM or less for very long.
Mammalian cells have developed systems that regulate the free Ca
2+
i
concentration
in the cell by extruding or sequestering the ion away from the cytosol. Along with
their other critical cellular functions, certain organelles, such as the mitochondria
and endoplasmic reticulum (ER), serve as high capacity storage sites for Ca
2+
ions.
Under the correct conditions, these stores can be transiently released into the cytosol,
resulting in a brief pulse of elevated Ca
2+
i
. This pulse serves to activate other signal
transduction machinery, which leads ultimately to an immediate cellular response, such
as degranulation or contractility, or changes in gene expression that result in altered
cell phenotype and function more slowly. Relaxation of the Ca
2+
i
signal is achieved by
the activation of the various sequestration and extrusion mechanisms operative within
the cell that serve to rapidly reduce the concentration of elevated free Ca
2+
to the
non-toxic level of approximately 100 nM. Other events besides Ca
2+
i
mobilization can be
measured by ow cytometry and also show transient kinetic behaviors. These include
membrane potential changes, pH
i
changes, Na
+
i
and K
+
i
ion changes.
94
Guide to Flow Cytometry
Measurement of Kinetic Events
This chapter will focus on one example of kinetic events available to all users, Ca
2+
i

mobilization, but other signal transduction pathways and events can be studied in kinetic
mode since the correct uorescent tools, such as pH and membrane potential sensors,
are available. Ca
2+
i
mobilization can be elicited by a number of different pathways
including the action of inositol trisphosphate (IP3) at the IP3 receptor on the ER store
or the activation of plasma membrane Ca
2+
channels. Here, the focus is on activation
of the phospholipase C (PLC) pathway, which can occur via activation of Gq type G
protein-coupled receptors (GPCRs) in which the Gq protein directly activates PLC
or via activation of tyrosine kinase pathways that can also activate PLC activity.
2
The
result of both pathways is the rapid hydrolysis of the plasma membrane phospholipid,
phosphatidylinositol trisphosphate (PIP2), to yield diacylglycerol (DAG) and IP3. The
released IP3 freely diffuses through the cytosol, binds to the IP3 receptor on the ER,
which functions as a Ca
2+
channel, and results in the release of Ca
2+
from the ER store
into the cytosol. The rate of release of Ca
2+
from the ER is typically sufcient to raise the
Ca
2+
i
from approximately 70-100 nM at rest to peak levels of 200-700 nM. As the initiating
signal transduction cascade wanes and the formation of IP3 declines, energy dependent
Ca
2+
pumps on the ER, the mitochondria and the plasma membrane will engage and
remove the Ca
2+
from the cytosol. Typically the Ca
2+
i
will be returned to resting levels
within less than one to two minutes of the initiation of the response. A Ca
2+
inux
pathway (SOCC or ICRAC) is activated when the ER store of Ca
2+
is reduced, but such
a mechanism is beyond the scope of this review and may be studied elsewhere.
3

Ca
2+
i
mobilization can be studied on most ow cytometers, as long as the data
acquisition software permits continuous acquisition and display of events over time so
that a continuous distribution of the status of the cells can be displayed during the time
kinetic measurement. The ready availability of uorescent Ca
2+
i
-reporting probes that
are optimally excited by either visible or ultraviolet wavelength lasers and the ease of
loading of these probes into viable cells makes them simple to use. The most accurate
dye for common applications is the ratiometric probe indo1, since the emission intensities
of the probe measured at two distinct regions of the spectrum will change inversely as
the Ca
2+
i
increases or decreases (Figure 1). By calculating the ratio of the two different
emission intensities, any variation in the extent of dye loading amongst different cells is
eliminated, and a very narrow range of Ca
2+
i
is indicated in most resting cell populations.
Calculation of the ratio of the uorescence intensity at two different spectral intensities
(e.g. 510 nm/410 nm) will give a value that increases or decreases as the Ca
2+
i
increases
or decreases over time, respectively. The drawback with indo1 is that it requires a
UV excitation source, such as a HeCd laser (325 nm), to be excited. Although not
ratiometric, several good alternative probes, such as uo-4 and Oregon Green 488
BAPTA-1, are available and provide nearly as reliable and accurate indices of Ca
2+
i
. Both
are as convenient to measure as standard uorescein-labeled probes since both are
excited by the 488 nm emitting argon lasers available as stock equipment on almost all
ow cytometers and both have peak emissions in the 515 nm to 535 nm range. In this
case emissions at a single wavelength region are acquired, and the emission intensity
increases or decreases as the Ca
2+
i
increases or decreases, respectively.
95
Kinetics
Figure 1. A bivariate distribution plot of cells loaded with indo1 showing the emissions at 400420 nm (x-axis) and
500530 nm (y-axis). The upper and lower populations are resting and stimulated cells, respectively. Data was
collected on a Dako MoFlo

using Summit Software.

An example of a Ca
2+
i
response measured by ow cytometry is shown in Figure 2. Here,
HEK-293 cells were loaded with indo1 and sampled from a standard tube containing
0.5 mL of the suspension. Carbachol (10 M) was added to activate the muscarinic
receptor constitutively expressed by the cells. The tube contents were gently mixed and
placed on the cytometer sample input. The sample was briey boosted to accelerate
transit of the cells to the laser intercept, and the data was acquired for three minutes.
Since a GPCR-mediated Ca
2+
mobilization response can develop within seconds, it is
important to minimize the time required to mix the sample and begin analysis. Otherwise,
the early phase of the response may not be observed.
Figure 2. A time kinetic plot of a Ca
2+
i
mobilization response showing the indo-1 emissions ratio (y-axis) and time (x-
axis). Data was collected on a Dako MoFlo

using Summit Software.

96
Guide to Flow Cytometry
Measurement of Ca
2+
i
Response Kinetics
A standard Ca
2+
response kinetic experiment can be performed for most mammalian cells
using the following techniques, assuming that the cells express a receptor that couples
to Ca
2+
mobilization. The cells should be grown under optimal tissue culture conditions.
Cells that are overgrown or starving for nutrients will show blunted responses, or none at
all. The cells should be harvested using normal techniques for tissue culture passage,
with special efforts not to treat the cells roughly (e.g. pipetting them too vigorously). To
load the cells with a Ca
2+
indicator, the probe should be in the membrane permeant,
ester form (e.g. indo1AM, uo4AM) and prepared in a stock solution of 1 mM in DMSO.
Stock solutions should be aliquoted and frozen at 20 C, thawed and used only once,
then discarded. The stock solution of probe should be diluted into the cell suspension
(1-5 x 10
6
cells/mL) to a nal concentration of 1-5 M. The cells should be in an isotonic
buffer such as Hanks Balanced Salts Solution (HBSS) containing Ca
2+
and Mg
2+
rather
than culture medium. Good loading occurs if the cell mixture is gently rocked on a
nutator or rotator for one hour at room temperature, but satisfactory loading also occurs
at 37C for 3040 minutes with occasional inversion to keep the cells suspended. The
best situation is to use the minimal amount of dye to load the cells to obtain the best
signal while loading sufcient dye to obtain a good signal.
After dye loading, the cells should be washed once and resuspended in HBSS at
0.55 x 10
6
cells/mL for analysis. Virtually all Ca
2+
i
mobilization events will be apparent
at room temperature. Aliquots of cells (0.51 mL) are sufcient for most experiments
running at normal ow rates. Every experiment should include a baseline measurement
where the cells are run for 1530 seconds with no additions to that sample. This
provides an index of the degree of homogeneity of the Ca
2+
i
levels in the population.
Ideally the population distribution will be very tight, but in some cases, distribution
of Ca
2+
i
levels is very diverse, and this can make appreciation of changes in Ca
2+
i

within the population difcult to resolve and interpret. The second important control
is a mock mixing test where the sample tube is removed from the instrument, buffer
control is added to the sample, the sample is mixed by gently swirling or icking the
tube, the sample is returned to the instrument and pressure is resumed with a sample
boost if necessary. This mock mixing control determines whether the cell population is
susceptible to mechanical shear forces, such as mixing and sample pressurization, that
can activate pressure sensitive ion ux mechanisms present in some cell lines. If this
control is not run, then a ligand can often be misinterpreted as stimulatory when in fact
the apparent response was simply due to the false response of the cells to the shear
forces encountered during ligand addition, mixing and sample pressurization. If such
activities are present, then efforts can be taken to reduce the vigor of the mixing and
pressurization steps. If one is quick and clear, these controls can be performed with the
same sample that will be exposed to ligand, and a continuous acquistion data le can
be obtained from a 1 mL sample over 2 minutes. When the ligand has been added, most
receptor-initiated responses with a moderate to high afnity ligand will develop within
5-30 seconds (Figure 2). Peak responses should be seen within 10-60 seconds. This
will be followed by a decline phase where the population shows a decreasing number
97
Kinetics
of cells with elevated Ca
2+
i
levels, and the average level of the population decreases.
The Ca
2+
ionophore, ionomycin (1-20 M), serves as an excellent positive control to test
whether the cells are properly loaded with reporter dye and whether the ow cytometry
system is properly set up.
It is important to note that, unlike microscopic analyses where the response of each cell
is evaluated over time, ow cytometry samples individual cells from a large population at
very discrete time points. Thus, kinetic ow cytometry data is a continuous sampling of
individual cells from a large population at single brief time points throughout the kinetic
measurement time span. This example clearly shows the response heterogeneity of the
population and the transience of the response.
References
1. Tsien RY. Intracellular signal transduction in four dimensions: from molecular design to
physiology. Am J Physiol 1992; 263(4 Pt 1): C723.
2. Taylor CW. Controlling calcium entry. Cell 2002; 111(6): 767.
3. Parekh AB. Store-operated Ca
2+
entry: dynamic interplay between endoplasmic
reticulum, mitochondria and plasma membrane. J Physiol 2003; 547(Pt 2): 333.