Take control

of your live cell assays

eBook

FEATURING SPARK® CYTO

LIVE CELL IMAGING SYSTEM
Take control
of your live cell assays

The need for more physiologically relevant and timely results from cell-based assays
continues to be a major challenge.

The use of increasingly complex assays and more advanced cellular models is placing
greater demands on the readouts and detection methods required to study them.

In this ebook we explore what it takes to tame automated cell imaging assays and take
back control of kinetic experiments to get reliable results more quickly, with fewer
errors, and less aggravation.

Contents
Live cell imaging empowers exploration 4
Six reasons live cell experiments can get out of hand 5-6
How to take back control of your live cell experiments 7-8
Real-time image Cytometry in a plate reader: Capturing the whole story 10-12
Monitoring cell migration and wound healing  13-17
Real Time Experimental Control (REC™) for cell-based assays 18-22
Spark® Motion. Automated cell analysis on your bench 23
The best
detect what
the rest ignore

IT’S TIME TO TAKE ANOTHER LOOK AT YOUR MICROPLATE READER.

Today’s scientific breakthroughs require technologies that let you see life’s
finer details without limits. Our microplate readers help you uncover deeper
insights into the biology of cells, with high reproducibility and scalable
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solutions to fit your team’s research.

www.tecan.com/rethink-your-reader
Spark® multimode reader is For Research Use Only. Not for use in diagnostic procedures.
© 2020, Tecan Trading AG, Switzerland, all rights reserved.
 Live cell imaging
empowers exploration

Image captured with Tecan Spark® Cyto of A431 cells cultured in a 96-well plate, acquired with the 10x objective, showing an overlay
of the blue, green and red channels.

Live cell imaging is one of the most important

techniques in the life sciences today. But behind
every great imaging assay, pity the poor scientist
grappling with the demands of biological
variability and complex kinetic cell assays. Live
cell experiments are often synonymous with
unsociable working hours, tedious protocols and
unrepeatable results.

Real-time cytometry is empowering life

science exploration
The popularity of imaging living cells continues to
grow year over year. In the last five years alone,
there have been nearly 8,000 scientific publications
on live cell imaging—more than in the preceding
40 years combined. (Source: PubMed Database). The number of publications on live cell imaging continues to rise
Why? Because the old adage “seeing is believing” each year. (source)
is actually true: images convincingly capture
aspects of living cells’ behavior and function that
are otherwise difficult or impossible to detect.
modalities, including both top- and bottom-read
Today’s cell imaging platforms enable real-time configurations, enable labs to multiplex more types
capture of individual cellular events in non- of assays and make use of novel fluorescent probes
destructive multi-well formats that support higher to quantify complex, dynamic events.
throughputs and robust, cell-by-cell, statistical
analyses. As your assays become more complex, so does
the potential for error. Read “Six reasons live
When image-based cytometry is combined with cell experiments get out of hand,” for common
the capabilities of a multimode plate reader, even challenges that we have seen with labs who are
more insights are possible. Multiple detection working with live cells.

4
 Six reasons
live cell experiments
can get out of hand
Real-time live-cell assays often involve sensitive
cell types and complex protocols that can easily
go awry—wasting valuable resources and possibly
giving you misleading results.
Read about six common pitfalls that can send your
live cell experiments spinning out of control.
1. Starting with unhappy cells.
Many live cell assays are doomed to fail before
they even start because the cells are not in the
exponential growth phase or have been subjected
to stress prior to the experiment—for example,
when transferring from the tissue culture incubator
to a slightly different environment in the imager or
plate reader.
Check out our blogs for more information about
the influence of confluence and environmental
conditions on live cell imaging experiments, and
what you can do to keep your cells happy when
preparing for your assay.
2. Experimental complexity.
Kinetic live cell assays can get very complex, very
fast. That’s because you need to factor in many
different fluorescent probes, time points,
dose concentrations, replicates, control wells, cell
types, and so on.
The more elaborate the experiment, the more
chances there are for variability to creep in and
mistakes to be made. To make matters worse,
manual steps and complicated instrument
control software can significantly increase the
likelihood of human error.
Unfortunately, some experimental complexity
is often unavoidable—you really do need all
those concentrations, replicates and time points!
Fortunately, cell imaging platforms are advancing
to help us accommodate the necessary complexity,
rather than avoid it. Read on to find out how.
3. Failing to adapt to biological variability.
Living cells are inherently variable, which means
that to obtain consistent assay results from day-
to-day or lab-to-lab, you may need to adjust
experimental protocols and timings on the fly.
For example, rather than always starting your
assay a fixed number of hours after seeding,
you may get more consistent results if you wait
a variable time period until the cells reach their
optimal confluence—say, 80%.
But what do you do if the cells reach 80%
confluence when you aren’t in the lab? Quite often,
we compromise and start the assay at the wrong
confluence rather than go back to the drawing
board and re-work the timings.
When you compromise protocol timings to fit your
schedule, the assay may still work, but reviewers may
question whether the results are biologically relevant.
5
In the example below, we see how variable factors imaging experiment may generate thousands—or
like percentage cell viability and fluorescent signal even hundreds of thousands—of images per run.
may dictate the most appropriate times to add Data storage and archiving can be expensive, and
various stimuli or probes, or when to start and stop servers quickly become filled to capacity.
imaging to ensure you capture the full response.
If storage space runs out in the middle of a critical
4. Disturbances and disruptions. run, you risk losing the cells and the entire data
Mechanical or environmental disturbances can set—a big toll in terms of the cost, time and labor
trigger cellular stress responses that will alter cell you put into your experiment.
behaviors and responses to stimuli, and may even For all these reasons, limiting the number of images
activate cell death pathways. Common culprits you need to acquire can be a smart move.
include lid-lifting, harsh addition of reagents or
compounds, wash steps, and transfer of plates from
one device to another.
Take back control with Spark® Cyto
5. Scale-up and miniaturization.
Kinetic assays may work fine at the prototype
stage, only to fall prey to mistakes when scale-
up suddenly amplifies the number of replicates,
pipetting steps, samples and compounds.
In addition, scale-up often entails miniaturization,
which means dealing with very small liquid volumes.
In such cases, evaporation—and by extension, assay
duration—becomes a significant concern. At the
same time, liquid handling accuracy and precision
become even more critical, and the chances of
mistakes and cross-contamination are further
magnified.

6. Image management challenges. Video: Revolutionize your cell experiments to never miss a
Depending on the assay complexity, a live cell critical biological event.

6
 How to take back
control of your
live cell experiments
Let’s take a look at some improvements we can
make to gain more control and eliminate error.
Improvement 1: Get onboard environmental
control.
At the start of the experiment, cells are seeded
into a 384-well plate, and left to incubate until
they have recovered and reached 80% confluence.
Crucially, you need to avoid shocking the cells
when transferring them from the tissue culture
incubator to the liquid handling station or detection
system. This means providing the cells with a stable,
onboard, humidified environment (typically 37oC,
5%CO2), where they can equilibrate for a sufficient
amount of time before compound is added and
measurements are taken.
Improvement 2: Automatically trigger
actions using thresholds.
Depending on the health of the cells at the time
of seeding, the length of time needed to reach
80% confluence can vary unpredictably. To avoid
the hassle of having to adapt your protocol and
working hours to suit the cells, the smart solution is
to automate confluence determination, and set up a
confluence threshold that will automatically trigger
subsequent steps.
An automated multimode reader and cell imaging
platform with this sort of conditional real-time
experimental control can minimize the amount
of manual intervention needed, while eliminating
subjective assessments — triggering additions
and reads at the right times, based on accurate
quantitative measurements.
Improvement 3: Reduce the number
of images acquired.
As shown in the schematic, the assay is configured
to run for approximately 48 hours from start to
finish. If we run the assay in just one 384-well plate,
collecting a single whole-well image in both red and
green channels
every hour from the point that the dyes have been
equilibrated (i.e., hours 17 through 48), we’ll need to
collect a jaw-dropping 24,576 images.
Can we do better?
Of course. It turns out that we are collecting many
images that are not needed, namely those at the
very top and bottom of the dose-response curves.
The key to reducing the image number is to use
threshold-based conditional programming to
collect only the images that are actually needed to
generate a reliable curve.
Here’s how it works:
After the cells have been equilibrated with dye mix,
we start continuously recording PI intensity using
a bottom read (no images required!). We can then
automatically detect an increase in fluorescence
when dead cells first start appearing, and use an
7
 As mentioned earlier, the more manual steps you
can eliminate the better.
Automating as much of the assay protocol as
possible will significantly improve accuracy,
decrease variability, reduce the chances of error,
and free valuable staff up for more important, less
tedious tasks.

The answer? A multimode imager with

Cell viability assay.
intensity threshold to trigger the 2-color imaging
to start from that point onward.
Similarly, if we can calculate percent viability by
analyzing the image data in real-time, we can set
second threshold which will trigger the imaging to
stop when viability bottoms out, in this case at 10%.
With both thresholds applied, imaging is carried
only from hours 20 through 44.
real-time experimental control
The best way to achieve hands-free, error-proof
kinetic cell assays is with a multimode reader that
includes onboard environmental control and lets
you perform image-based confluence assessment,
intensity measurements, fluorescence imaging and
real-time image analysis — all on the same system.
And crucially, you need a system that allows you to
set up threshold-based conditional responses easily
and with no need for programming skills.
Cell population analysis demands the full picture

The result
Automated real-time experimental control reduces
the number of acquired images by 5376 — over
20%! That’s a major savings in terms of both
experiment time and storage space.

Improvement 4: Create a “walk-away”

process.
A final consideration is the amount of hands-on
time needed to run the assay from start to finish. Webinar: Never Miss a Critical Biological Event.

8
The best detect
better ways to
accelerate discovery

THE SPARK® MULTIMODE MICROPLATE READER PLATFORM

ENABLES YOU TO DO MORE.
The use of increasingly complex assays and more advanced cellular
models is placing greater demands on the readouts and detection
methods required to study them. Spark gives biologists the flexibility
to seamlessly go from a quick visual check to a complex cytotoxicity
or gene expression study using a single instrument.

Learn how Tecan’s Spark platform allows you to uncover new possibilities
for your cell-based assays.
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www.tecan.com/rethink-your-reader
Spark® multimode reader is For Research Use Only. Not for use in diagnostic procedures.
© 2020, Tecan Trading AG, Switzerland, all rights reserved.
 Real-time image
Cytometry in a plate reader:
Capturing the whole story
Cell-based assays have a variety of practical
uses within scientific research and are an integral
component of many scientists’ workflows from
testing tumor resistance for cancer research to
helping identify novel therapeutic compounds in
drug discovery. They are often used as an in vitro
tool to measure the number of cells at different
time points throughout an experiment. For
example, if a scientist is testing the efficacy of a
novel compound on eradicating cancer cells, a
plate reader can be used to quantify the number
of living cells before and after administration
of the compound. Conventional assays use a
derived fluorescent signal to measure enzymatic
activity as an indicator of cell viability such as
adenosine triphosphate (ATP) and tetrazolium/
resazurin reduction1.
Cell-based assays can also be used to measure:
• Receptor binding
• Gene expression
• Organelle function
• DNA synthesis
• Number of cell divisions in a population2
There are, however, many limitations to using
conventional in vitro cell-based assays which can
compromise experiments and ultimately impact
the clinical and translational potential of cell-
based work. These include the following:
Maintaining optimal physiological conditions
To accurately measure the number of viable
cells during an experiment, it is imperative to
maintain optimal physiological conditions. If any
component of the extracellular medium is altered
accidentally, cell viability can change without
the scientist’s knowledge and independent of
intentional experimental manipulations.
General biological variability
Different cell types have variable dynamics in
cell proliferation and other biological processes
which can confound the reproducibility of
experiments.
Time and cost-intensive
These assays require multiple rounds of
experiments, continuous monitoring, and
manual handling by the scientist - which greatly
increases the risk of error and the likelihood that
the experiment will need to be replicated.
Importance of live-cell analysis
Recent advances in cell-based technology
mitigates some of these roadblocks by utilizing
real-time and automated imaging software.
The main advantage of automated imaging is
10
that it eliminates the need for user intervention,
which helps ensure that biological samples
remain intact and physiological conditions
remain constant throughout experiments.
Perhaps most important, novel platforms
consider the complexity of biological processes
by cytometry, which not only measures cell
survival, but also cell morphology, size, and
cell cycle phase. Detecting parameters such
as these can be extremely informative, for
example, in measuring cancer metastasis3. In
addition, large cell populations usually have
significant heterogeneity in gene expression, and
the sensitivity of automated real-time
imaging platforms can capture this heterogeneity
to give scientists a much more comprehensive
analysis4.
Introducing Spark® Cyto technology
Tecan’s new Spark Cyto technology
revolutionizes in vitro cell viability assays by
semi-automating traditionally labor-intensive
experiments. The technology is equipped with
multimodal detection software and fluorescent
imaging technology to allow for simultaneous
visual and functional measurements. Researchers
can cleanly record the entire area of a 96- or
384-well microplate in one single image (Figure
1) without tilting or distortion, so that they
never miss a crucial moment or component of
the cell population. Being able to capture a full
Figure 1. Single image of a complete well reveals the
distribution of dead (magenta) and viable (green) HeLa cells.
well with a single image provides researchers
with the whole story. With three magnification
levels and four channels for fluorescent and
bright field imaging capabilities, researchers
can be confident in the quality of their cell
analysis. Spark Cyto has LED-based illumination
with a reliable autofocusing system giving the
experimenter confidence that they can walk away
from long experiments5.
Additionally, optimal conditions are maintained
during experiments due to several automated
parameters. To reduce the risk of sample
contamination and eliminate the need for
manual intervention, the Spark Cyto has uniform
temperature control of up to 42 °C, dynamic gas
control for CO2 and O2, and a humidity cassette
with the patented Lid Lifter to reduce evaporation
in the microtiter plate.
The sensitivity of the Spark Cyto for standard
detection modes enables it to measure DNA,
RNA, and ELISA results5. This is important
as routine assays fail to acknowledge the
complexity of human pathophysiology, for
example cancer metastasis, and only focus on
physical translocation of cells4.
Malfunction in cellular processes, and even
healthy biology, is also extremely intricate
therefore it is crucial for scientists to be able to
extract a large amount of information from their
cell population aside from merely cell survival.
To help decrease the workload of experimental
setup, the SparkControl software includes
several predefined protocols. For example, the
cell viability application utilizes calcein-AM and
propidium iodide (PI), to detect viable and non-
viable cells, respectively.5
Key cytometry applications
Scientists can use Spark Cyto for many common
cytometric assays, including:
• Confluence
Confluence, or the percentage of cells
covering the surface of a plate, is measured
automatically with the bright field detection
setting and is displayed in yellow so that
the experimenter can easily visualize the
results.
11
• Nuclei Counting
Nuclei counting is a crucial measurement
used to detect the number of cells in a
population, and Spark Cyto has a feature
specifically optimized for counting nuclei
labeled with blue fluorescent dye such as
Hoechst 33342 or DAPI.
• Transfection Efficiency
Transfection efficiency determines how many
cells have taken up and expressed
a gene of interest (i.e., fluorescent protein),
or whether a tumor biomarker has been
overexpressed.
• Cell Viability
The preset cell viability application, which
has been briefly described above, can quickly
distinguish between live and dead cells using
two different fluorescent dyes.
• Cell Death
It is crucial in many experiments to measure
certain types of cell death, such as necrosis
and apoptosis, and Spark Cyto can detect
each of these processes using Propidium
iodide and Annexin V-FITC for necrosis and
apoptosis, respectively6. Certain cancer
therapeutics aim to induce apoptotic cell
death, and certain vascular injuries cause
necrotic cell death, so it is vital information
to seamlessly distinguish between the two.
These features can be accessed by a single
click of an application.
SparkControl software
In addition to stabilizing the physiological
environment, some of the greatest challenges
that scientists face during experiments include
sample contamination and manual error. These
can be mitigated by SparkControl software.
Perhaps most captivating is SparkControl
software’s ability to automate long-term kinetic
assays, providing scientists with a hands-off
solution for complex experiments. For example,
if an experiment requires the addition of a certain
reagent at a specific time point, SparkControl allows
this to be triggered automatically. The reagent
dispenser (Spark injector) has heating and stirring
options to minimize perturbation of the system
following addition of the reagent6.
To quickly and easily detect changes in
fluorescent expression of cells in the population,
the 3D scanning capabilities allow simultaneous
excitation and emission scans. Using Spark
Cyto’s extended dynamic range, experimenters
can be sure that even a very low signal of
fluorescent expression will be detected. In
addition, researchers have the flexibility to use
the SparkControl software solely for its imaging
capabilities, or for both imaging and detection
analysis6.
Spark Cyto brings many advancements to the
world of in vitro cell-based assays by utilizing
innovative imaging technology and maintaining
pristine experimental conditions to ensure
reproducibility and validity of experiments.
It is now possible to detect and analyze an
unprecedented range of biological processes in
a single experiment. These major improvements
from traditional cell-based assays may be the
impetus that will propel scientists to uncover
many cutting-edge discoveries.
References
1. Riss, T. L., et al. (2004). Cell Viability Assays. Assay
Guidance Manual.
2. Gordon, J. L., et al. (2018). “Cell-Based Methods for
Determination of Efficacy for Candidate Therapeutics in
the Clinical Management of Cancer.” Diseases 6(4).
3. Barteneva, N. S., et al. (2012). “Imaging flow cytometry:
coping with heterogeneity in biological systems.” J
Histochem Cytochem 60(10): 723-733.
4. Varankar, Sagar S, and Sharmila A Bapat. “Uncoupling
Traditional Functionalities of Metastasis: The Parting of
Ways with Real-Time Assays.” Journal of clinical
medicine vol. 8,7 941. 28 Jun. 2019, doi:10.3390/
jcm8070941
5. Eggenhoffer, Nicole. Real-time experimental control for
cell-based assays (2019) Tecan, Austria.
6. Tecan. (2019) Spark Cyto: Live Cell Plate Reader with Real
Time Cytometry. Brochure available from here [Accessed:
September 23, 2019]
12
 Monitoring cell migration
and wound healing 
CELL IMAGING WITH THE SPARK® MULTIMODE READER PLATFORM
INTRODUCTION
Cell migration is a central process in the
development and maintenance of multicellular
organisms, with tissue formation during embryonic
development, wound healing and immune
responses all requiring the orchestrated movement
and assembly of cells. Abnormalities in cell
migration are associated with various diseases –
including cancer, atherosclerosis and arthritis – and
so understanding cell migration and the biological
events that trigger the movement of cells from
one location to the other is a key focus of cell-
based research.
In this context, imaging-based analysis methods
are highly valuable tools for the investigation
of cell migration.
While image analysis using visual microscopy is a
long-standing and reliable method in cell biology, it
is very time-consuming and laborious. Automated
imaging and analysis of cell samples greatly
facilitate the experimental workflow and increases
the throughput while minimizing experiment-to-
experiment variations caused by variable starting
conditions. Live imaging-based readouts can be
used for walkaway monitoring of cell growth and
health during long-term experiments.
Tecan’s Spark instrument platform has an integrated
bright-field cell imaging module that enables label-
free and real-time assessment of cell confluence
in microplate wells. The reader is able to detect
cell-covered areas in 6- to 96-well plate formats
and calculate the relative confluence ratio based
on the analysed area. The confluence ratio can also
be used to normalize other cell-associated signals
(e.g. the cellular ATP content) to the number of
cells in the sample. The Spark’s confluence-imaging
function allows the study of various different
forms of cell migration – such as chemotaxis,
transmigration and tissue invasion, as well as the
analysis of the metastatic potential of tumor cells.
The Radius™ 96-well Cell Migration Assay is a
so-called ‘gap-closure’ assay system that uses
a defined cell-free area that can be repopulated
by cells and monitored in real-time by visual or
automated microscopy. Unlike Boyden chamber
assays which can only be analysed at endpoint, the
Radius assay uses proprietary cell culture plates
with a carefully-defined, circular spot created with
biocompatible hydrogel (Radius gel) at the center
of each well. When cells are seeded in the well, they
will attach everywhere except the gel spot, creating
a cell-free zone. Following cell seeding the gel spot
is removed, allowing migratory cells to move across
the area and close the gap. This ‘artificial wound’
in the cell monolayer can be used to study cell
migration, cell proliferation and wound closure. Due
to the gap’s well-defined area and position inside
the well, this method is a much more consistent
alternative to conventional scratch assays.
This application note describes the application of
13
the imaging capabilities of the Spark multimode
readers for the monitoring of cell migration in the
context of wound healing, using the Radius 96-well
Cell Migration Assay as a test system.
Furthermore, this note describes the correlation
between cellular confluence and intracellular
ATP content as an indicator of cell viability and
metabolism.
MATERIALS AND METHODS
Migration
The Radius 96-well Cell Migration Assay (Cell
Biolabs, CBA-126) was tested in combination
with the confluence imaging function of a Spark
multimode reader to monitor the cell migration
and repopulation of the cell-free area over a
period of 30h. The kit was used according to the
manufacturer’s instructions [1]. Two biliary tract
cancer cell lines (“CL1” and “CL2”) were used. CL1
is known to have negligible migration properties,
whereas CL2 is known to show high migration
activity. The cells were cultured in DMEM high
glucose supplemented with L-glutamine, penicillin/
streptomycin, sodium pyruvate and HEPES +
10% fetal calf serum (FCS). For the migration
experiments, the cells were transferred into FCS-
free medium to minimize cell division and be able
to monitor only migration and wound healing
activity instead. The cells were then seeded into
transparent Radius 96-well Cell Migration Assay
microplates (supplied with the kit) at an initial
density of 3x104 cells/well (the optimal seeding
density was predetermined) and left to adhere
over night.
The inserts of the Radius plates were removed
at the beginning of the experiments, and the
repopulation of the cell-free area by migration cells
was monitored over a period of 30 hours.
The migration assay was performed in two ways:
a manual and an automated/kinetic setup.
1. Manual: The test plate was kept inside a
conventional CO2 incubator between the
measurements to provide a suitable temperature
and gas atmosphere, and transferred to the
instrument for the readouts at 0, 2, 4, 6, 8, 24
and 30h.
2. Automated: The entire measurement was
performed inside the Spark reader, using the
instrument’s environmental control features to
create an automated workflow and maintain
5% CO2 and 37°C throughout the entire
measurement period.
Parameter Setting
Measurement Cell Confluence
Readout mode Kinetic
Duration 60 cycles
Interval 30 min
Pattern central
Settle time 0 ms
Data analysis activated
Well border detection none
Table 1. Measurement settings for cell migration experiments.
The measurement settings for the automated/
kinetic cell migration experiments are summarized
in table 1. In the manual setup, each measurement
was performed in endpoint mode instead
of kinetic mode. For the automated/kinetic
setup, the confluence was recorded over the
entire experimental period of 30 hours, with
one measurement point every 30 minutes. The
confluence pattern was set to ‘Central’, meaning
that a central picture from the center of each well
was taken and analyzed. Importantly, the ‘Central’
pattern is only useful when the area of interest, i.e.
the cell-free spot, is located in the middle of the
well. The measurement/imaging area can
not be repositioned within the well, and the
detected confluence value only reflects the
percentage of cell-covered area in the analysed
spot(s). The confluence values calculated based on
the pictures were then evaluated as an inversely
proportional indicator of cell migration, with the
initial (0h) value assumed to represent a 100% cell-
free area.
Correlation of confluence
and cellular ATP content
The second aim of this study was to correlate the
confluence signals with the ATP content of the
cells. In this context, the CellTiter-Glo® Luminescent
Cell Viability Assay (Promega, G7571) was used to
detect and quantify the ATP levels in the cells. The
assay is a homogeneous method of determining
the number of viable cells in culture based on
14
quantitation of their ATP level, as an indicator of
metabolically active, viable cells present in the
sample [2].
Again, two biliary tract cancer cell lines (“CL3”
and “CL4”) were used and seeded into 96-well
plates at a range of initial cell numbers per well
(25000, 12500, 6750, 3125 and 1563 cells/well).
After 24, 30, 48 and 50h, the confluence in each
well was measured using the ‘Whole well’ pattern,
immediately followed by the ATP quantification
assay in the same wells. The results were evaluated
by calculating the luminescence/confluence ratio,
with outliers identified and removed based on the
Grubbs outlier detection test.
RESULTS
Migration / Wound Healing
The expected migration pattern of the two
different cell lines could be clearly seen using the
confluence function of the Spark reader (figure 1).
While CL1 showed no or only negligible migration,
CL2 exhibited continuous repopulation of the cell-
free area over the analysis period.
While the migration behavior over the total
analysis period shows similar dynamics for
manual and automated handling, the latter has
the advantage that no data points are lost due to
overnight gaps where the operator is not present
to perform the manual plate handling. Automated
handling therefore provides consistent and gap-
free analysis of the cells’ migration dynamics.

% cell-
% cell-
free
Hours Av. conf. SD free
area
area
(norm.)
0 82.7 1.15 17.3 100
6 82.8 1.15 17.2 99
11 85.3 1.15 14.7 84
15 87.0 1.73 13.0 75
18 88.0 2.00 12.0 69
22 89.4 2.52 10.6 61
27 91.3 2.52 8.7 50
30 92.3 3.21 7.7 44

Table 2. Confluence / migration dynamics and calculated cell-

free area of CL2 (selected data points).

The continuous repopulation of the cell-free area

Figure 1. Dynamics of cell-free area repopulation by CL1 and CL2
(shown as percentage of cell-free area), comparing manual vs.
automated handling of both cell lines..
can be clearly seen in figure 2 where the time
course of gap closure is documented. Over the
30h analysis period, repopulation of more than half
of the initial cell-free area could be observed.
The cell-free area was observed to get
progressively smaller with increasing cell migration
into the circle.
In figures 1 and 2, the initial value (0h) was assumed
to represent 100% cell-free area. All other values

15
Figure 2. Gap closure by CL2 (biliary tract cancer cell line with high migrating potential) over 30h.
are given in relation to the initial situation (also see
table 2).
The extent of migration was determined by
plotting the inverse confluence value detected
by the SparkControl™ software in the selected
measurement area, i.e. the single picture in the well
center (figure 2). For an exact determination of
the gap area, the use of third-party image analysis
software, such as ImageJ, is recommended.
Correlation of confluence and cellular
ATP content
Figure 3 shows the correlation between confluence
and ATP-based luminescence over a period of
54h using different initial cell numbers at the time
of seeding. Both signals exhibited similar dynamics
over time, indicating the progressive growth of the
cells which is characterized by both an increase of
the cellular ATP content and increasing cell density
on the well bottom. With cell numbers below 3000
cells/well the confluence values were outside of the
recommended range (<10%), so it is advisable to
use higher cell numbers for reliable experiments.
From 3125 cells/well upwards a good correlation
between ATP signal and confluence values could be
observed, indicating that it is possible to normalize
the results using confluence measurements instead
of the more laborious cell number determination by
measuring the ATP content.
CONCLUSION
The results of this study show that the
Spark reader platform is ideally suited to the
measurement of cell migration and wound
healing assays such as the Radius 96-well Cell
Migration Assay.
The instrument’s brightfield imaging-based
confluence function enables the high-quality and
label-free real-time monitoring of cell movement,
allowing the dynamics of gap closure and wound
healing to be quantified and followed over time.
The reader’s SparkControl software makes it easy
to quantify cell confluence right in the microplate
wells, with user-selectable patterns for analysis of
different areas within the well and automated well
border detection for any type of microplate.
The ability to correlate confluence with cellular
ATP content further simplifies cell-based assay
workflows by allowing complete walk-away
automation of experiments.
As the confluence measurement is label-free
and does not interfere with any other cellular
characteristics or properties, it can be performed
continuously over the entire experimental period,
providing a simple and reliable alternative to more
traditional, costly and/or laborious techniques for
the determination of the population size/mass of
cells of interest.
16
Figure 3. Correlation of confluence and ATP-based luminescence signals at different cell numbers.

References CL cell line

1. Radius 96-Well Cell Migration Assay, Product Manual, DMEM Dulbecco’s modified Eagles medium
EM emission
CBA-126, Cell Biolabs Inc., San Diego, USA
EX excitation
2. CellTiter-Glo Luminescent Cell Viability Assay, Technical FCS fetal calf serum
Bulletin 288, 03/15, Promega, Madison, WI, USA GFP green fluorescent protein
PBS phosphate-buffered saline
ACKNOWLEDGEMENTS RFU relative fluorescent units
We would like to thank Dr Christian Mayr and Dr Tobias
SD standard deviation
Kiesslich from the Laboratory for Tumour Biology and
About the author
Experimental Therapies (TREAT), Institute of Physiology and Dr Katrin Flatscher is an application scientist at Tecan
Pathophysiology, Paracelsus Medical University (PMU) for Austria. She studied molecular biology at the University
performing the experiments and evaluating the data. of Salzburg and focused on cell biology and immunology
research during her PhD. She joined Tecan in 2007 and has
ABBREVIATIONS been involved in the development of the Infinite as well as
ATP adenosine triphosphate the Spark reader series.
399677 V2.0 01-2017

17
 Real Time Experimental
Control (REC™) for 
cell-based assays
WORKFLOW AUTOMATION WITH THE SPARK® CYTO MULTIMODE READER
INTRODUCTION
The Spark Cyto enables semi-automation of
workflows for cell culture experiments. The
combination of multimode detection and
fluorescence imaging capabilities makes it possible
to perform visual and functional examination of cell
samples in a single experiment.
The Spark Cyto is equipped with a high-tech
imaging module, incorporating three user-selectable
objectives, four fluorescence channels, LED-based
illumination and a rapid and highly reliable LED-
based autofocusing system. Operated via the
intuitive SparkControl™ software, the system offers
advanced features – such as full environmental
control and kinetic conditioning software– providing
a walkaway solution for long-term cell-based assays
and complex experimental set-ups.
Many live cell kinetic experiments require specific
actions at different points in the experiment – for
example, the addition of a compound once a certain
confluence is reached. Real Time Experimental
Control (REC) allows certain actions – such as
injection of reagents – to be triggered automatically
as part of a kinetic experiment, and offers real-time
data and image analysis for complete confidence in
your results.
This application note demonstrates how REC can
be used as part of a compound screening workflow,
showing how it is set up within SparkControl, and
the valuable information that can be obtained from
a single experiment.
MATERIALS AND METHODS
GFP-transfected HeLa cells (ATCC® Number: CCL-
2™) were cultured in RPMI 1640 medium (Gibco,
#31870074; supplemented with 10 % FBS) at 37
°C and 5 % CO2 in a humidified atmosphere. Cells
harvested with trypsin/EDTA were then counted to
determine exact cell numbers, and 15,000 cells/well
were seeded in 300 µl of medium in a 96-well, black
tissue culture plate (TEC96fb_cell_clear). The cells
were allowed to adhere before the unlidded plate
was placed into the Spark Cyto’s large Humidity
Cassette filled with 5.2 ml of deionized water per
reservoir. The reader’s injectors were rinsed with
70 % EtOH and deionized water, then primed with 1
% saponin solution (Roth, #4185, diluted in growth
medium).
Creating the method in SparkControl
The aim of this experiment was to monitor cell
growth with the Spark Cyto, and determine the
cytotoxic effects of different concentrations of
saponin over time as part of a semi-automated
workflow. In order to standardize the experiment,
the injection of saponin was conditionally
programmed to be carried out when the confluence
of a reference well reached the chosen level.
In brief, the method consisted of gas, humidity and
temperature control, and a kinetic loop consisting of
18
fluorescence imaging, bottom reading fluorescence Parameter Setting
(FI bottom) measurements and conditional saponin
Plate TEC96fb_cell clear
injection.
Gas control CO2 5 %
The plate definition was chosen and the whole Temperature 37 ˚C
plate was selected for measurement within the
predefined plate strip. In addition, the large Measurement mode Kinetic
Humidity Cassette and ‘No lid’ options were No. of cycles 24
selected (see Figure 1). Interval time 2h

Measurement mode Fluorescence imaging

Application User defined
Objective 4x
Pattern Whole-well
Border offset 50 µm
Bright field
Figure 1. Plate strip highlighting wells to be imaged, and the use
Focus offset 0 µm
of the Humidity Cassette. Sensitivity 50 %
4-30 µm width, 4-40 µm
Object size
length
The large Humidity Cassette containing the test Green channel
plate was placed in the instrument, and a kinetic LED intensity 100 %
measurement protocol was set up in Method Editor
Focus offset 0 µm
using the detection strips and settings shown in
Figure 2 and Table 1, respectively. Exposure time 40 ms
Sensitivity 70 %
4-30 µm width, 4-40 µm
Object size
length

Measurement mode FI bottom

Excitation 485 nm
Emission 535 nm
Gain Calculated from A1
Flash number 30
Integration time 40 µs

Table 1. Measurement settings.

FI bottom measurements were performed with the

gain and Z-value calculated from well A1 (untreated
control) using the Optimal Read function.

Figure 2. Collapse of all strips necessary for the kinetic
experiment, including conditional injection.
Environmental control settings were defined outside
of the kinetic loop, with CO2 concentration set to 5 %
and temperature to 37 °C. Within the kinetic loop
strip, 24 cycles, at 2 hour intervals, were defined.
A Condition strip leads to the execution of all
following (indented) strips. In this case, several
conditions are set, for the injection of different
volumes of the saponin working solution on
reaching a specified confluence level of >89 % in a
reference well (B5). Within each condition the box
‘Executed once’ was checked (see Figure 3).

19
 Figure 4. Shaking strip displaying settings for REC experiment.

were refilled with 5.2 ml of deionized water to ensure

optimal experimental conditions.

Figure 3. Condition and Part of Plate strips, including settings for
a single saponin injection.
Injection was then performed via an Injector strip. A
separate Condition, Part Of Plate and Injector strip
was created for each injection volume – 6, 9, 15, 20
and 30 µl – resulting in final concentrations of 0.02,
0.03, 0.05, 0.06 and 0.1 % saponin.
RESULTS
While the default sensitivity settings were chosen
for method execution (as shown in Table 1) post-
acquisition optimization to 83 % sensitivity for the
green fluorescence channel was performed using
Image Analyzer.
The results show that the green object count steadily
increases over time, until the confluence condition
was fulfilled at cycle 12 (Figure 5).

Table 2 shows the final plate layout for the test

plate indicating the injection volumes of 1 % saponin
solution and the final saponin concentrations in the
wells.

1 2 3 4 5 6 7 8 9 10 11 12
A
Untreated (no saponin injected)
B
C 0.02 % (6 μl of 1 % saponin solution injected)
D 0.03 % (9 μl of 1 % saponin solution injected)
E 0.05 % (15 μl of 1 % saponin solution injected)
Figure 5. Confluency mask and fluorescent image of B5 reference
F 0.06 % (20 μl of 1 % saponin solution injected)
well at cycle 12.
G
0.1 % (30 μl of 1 % saponin solution injected)
H

Table 2. Plate layout indicating the final saponin concentrations (%)

after injection.
The Shaking strip was used to ensure even
distribution of saponin within each wells. Shaking
was performed in situ for 60 seconds, using a
double orbital mode, an amplitude of 2.5 mm, and a
frequency of 108 rpm (see Figure 4).
The method was paused after 10 cycles, the wells
were replenished with 100 µl of fresh, prewarmed
medium, and the reservoirs of the Humidity Cassette
Following saponin injection, the green object count
steadily decreased in a concentration-dependent
manner in all treated wells, whereas untreated
controls showed an increase in green object count
until a plateau was reached at cycle 17 (see Figure 6).
All saponin concentrations led to a large decrease
in FI bottom signal (up to a max. signal of ~33,000
RFU, see Figure 7), confluence level (13 % for 0.06 %
saponin, see Figure 8) and almost complete loss of
green object count (Figure 6).
For 0.02 % saponin, the curve’s course was less

20
steep, meaning the cells lost viability more slowly, 100

demonstrating the concentration dependency of 90

80
saponin-induced cell death.

Confluence [%]
70
60
The slight increase of FI bottom signal for samples 50

treated with 0.03, 0.05, 0.06 and 0.1 % saponin can be 40

30
explained by the background signal of additional GFP 20
being released from dead or dying cells, which have 10
lost their membrane integrity. 0
0 5 10 15 20 25 30
Cycle #

Untreated 0.02 % saponin 0.03 % saponin

18000 0.05 % saponin 0.06 % saponin 0.1 % saponin

Number of green objects detected #

16000
14000
12000
Figure 8. Confluence level of HeLa GFP cells plotted against
10000
cycle number (total of 48 h, 2 h interval time, injection of saponin
8000
at cycle 12).
6000
4000
2000
0
0 5 10 15 20 25 30
-2000
-4000 CONCLUSIONS
Cycle #
The Spark Cyto multimode reader is ideally suited
Untreated 0.02 % saponin 0.03 % saponin
for semi-automated workflows combining live
0.05 % saponin 0.06 % saponin 0.1 % saponin
cell imaging with other detection modes. A single
experiment approach allows efficient generation of
Figure 6. Number of green objects detected, plotted against results from multiple detection modes, under the
cycle number (total of 48 h, 2 h interval time, injection of saponin same standard conditions and time points, eliminating
at cycle 12).
the need and expense of performing multiple
cellular assays in parallel. The result-dependent
Condition strip enables detection (eg. absorbance)
80000
or actions (eg. injection) to be carried out at the
70000
right time, without the user needing to be present
for subjective assessments, reducing restrictions on
FI bottom signal [RFU]

60000
when experiments can be performed and enhancing
50000
the reproducibility of results. Using the Spark Cyto’s
40000
advanced features, procedures can be standardized
30000
and productivity enhanced, allowing high quality
20000
reproducible results to be obtained every time.
10000

0
0 500 1000 1500 2000 2500 3000
ABBREVIATIONS
Time [min]
GFP green fluorescent protein
Untreated 0.02 % saponin 0.03 % saponin FI fluorescence intensity
0.05 % saponin 0.06 % saponin 0.1 % saponin LED light-emitting diode
REC real-time experimental control
Figure 7. GFP signal of HeLa GFP cells plotted against cycle number EDTA ethylenediaminetetraacetic acid
(total of 48 h, 2 h interval time, injection of saponin at cycle 12). EtOH ethanol
100 CO2 cardon dioxide
90
80
RFU relative fluorescence units
Confluence [%]

70
Complementary
60 detection modes, combining the ABOUT THE AUTHORS
area50 algorithm for bright field imaging and the Dr. Nicole Eggenhofer is an application specialist at Tecan
40
segmentation
30 algorithm for the green fluorescence Austria. She studied genetics at the University of Salzburg
channel,
20 provide corroborative results within and focused on cell biology and microbiology during her Ph.D.
a single
10
experiment. Nicole gained further experience in the field of molecular
0
0 5 10 15 20 25 30
Cycle #
21
Untreated 0.02 % saponin 0.03 % saponin

0.05 % saponin 0.06 % saponin 0.1 % saponin

biology before joining Tecan in 2017. She has been involved in
the development of Spark Cyto.
Dr. Katrin Flatscher is an application specialist at Tecan
Austria. She studied molecular biology at the University of
Salzburg and focused on cell biology and immunology
research during her PhD.
She joined Tecan in 2007 and has been involved in the
development of the Infinite as well as the Spark multimode
reader series.
Dr. Lynda O’Leary is an application specialist at Tecan Austria.
She received her PhD in Biochemistry at the National University
of Ireland, Galway, working in the field of apoptosis and cell
signaling. Following this, she joined the Institute of Cancer
Research in London as a postdoctoral fellow focusing on
metastatic dormancy in breast cancer. Lynda has extensive
experience in a wide range of molecular cell biology techniques,
cell culture and imaging. In 2018, she joined the sales and
marketing department with a primary focus on the Spark Cyto
multimode microplate reader.
401053 V1.0, 2019-07
22
 Spark® Motion. 100
90
80 Viable cells

Automated cell analysis

70
60

% cells
50
40 Apoptotic cells
30 Necrotic cells

on your bench
20
10
0
time

Spark Motion expands your live cell analysis capabilities. It combines the
Spark Cyto live cell imager and plate reader with a robotic arm and incubator
to offer walkaway automation, allowing reading, imaging and analysis 40 PLATE
of multiple plates under controlled conditions for several days. INCUBATOR

AUTOMATED
COMPLETE WALKAWAY AUTOMATION PLATE
FOR LIVE CELL EXPERIMENTS HANDLING

•A
 utomated cell incubation and analysis of up to 40 plates
increases throughput.
•M
 ultiplex analysis (eg. luminescence and imaging) provides
more insights and increases reproducibility.
PLATE
•P
 atented Lid Lifter™ technology protects plates outside the IMAGING/
incubator, avoiding the need for expensive safety cabinets. READING

COMPREHENSIVE data and analysis HIGHLY FLEXIBLE read modes

• W hole-well imaging of 96- and 384-well plates • A bsorbance
• Reading and imaging in the same experiment • Fluorescence (FP, TRF, TR-FRET)
• Image acquisition and data analysis in parallel, • Luminescence (single and multi-color)
for fast access to results • Alpha Technology

EASY-TO-USE predefined applications EXTENSIVE imaging capabilities

• C onfluence • 4 fluorescent colors (blue, green, red, far-red)
• C ell counting • B right field and digital phase contrast
• C ell viability • 3 magnifications (2x, 4x, 10x)
401625 V2.0, 2020-08

• C ell death • L abel-free autofocus

www.tecan.com/spark-motion
Spark Cyto and Spark Motion are For Research Use Only. Not for use in diagnostic procedures.
© 2020 Tecan Trading AG, Switzerland, all rights reserved. For disclaimer and trademarks please visit www.tecan.com.
23
Tecan – Who we are.
Tecan is a leading global provider of life science laboratory instruments for biopharmaceutical, forensic, clinical
diagnostic and academic markets, specializing in the development and production of automation and detection
solutions, including imaging and microplate readers, microarray products and washers.

Founded in Switzerland in 1980, Tecan has manufacturing and R&D sites in both North America and Europe,
and maintains a sales and service network in 52 countries. To date, Tecan has distributed approximately 20,000
microplate readers worldwide, and is committed to continuous technological improvements and compliance with
the highest global quality standards.

Learn More.
To learn more about Tecan Spark® Cyto imaging reader and our complete microplate portfolio,
visit https://www.tecan.com/rethink-your-reader.

For Research Use Only. Not for use in diagnostic procedures.

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Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication; however, it is possible that omissions or
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401717 V1.0, 2020-09

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© 2020, Tecan Trading AG, Switzerland, all rights reserved. For disclaimer and trademarks please visit www.tecan.com.