White Paper
Inducing hypoxia inside Tecan’s Infinite® 200 PRO multimode reader
Christian Oberdanner1, Andreas Eger2, Julia Hinteramskogler2, Nico Jacobi2,
Anita Koppensteiner2, Maren Pflüger2, Sarah Verna3, Gerald Probst1
1
Tecan Austria GmbH, Untersbergstraße 1A, Groedig, Austria
IMC Fachhochschule Krems, University of Applied Science Krems, Piaristengasse, Krems, Austria
3
Thermo Fisher Scientific, 75 Panorama Creek Drive, Rochester, NY, USA
2
Introduction
The present paper describes the capability of Tecan’s Gas
The novel enhanced version of the GCM allows the
Control Module (GCM™) for the Infinite 200 PRO multimode
simultaneous adjustment of CO2 (0-10 %) and O2 (0.1-21 %)
reader to induce hypoxia in cell-based systems by
levels, enabling a wide range of different experimental
simultaneous regulation of CO2 and O2 levels inside the
applications, including growth rate studies with various
reader’s measurement chamber.
facultative anaerobic organisms and hypoxia research.
Tecan’s innovative and patent pending GCM for the Infinite
Hypoxia, the depletion of oxygen in cells and tissues, plays a
200 PRO multimode reader series (Figure 1) is designed to
significant role in several pathological conditions such as
control the CO2 and the O2 levels inside the measurement
ischemia-related diseases (2), cancer (3-5) and aging (6). In
chamber of the reader. Tecan has previously demonstrated
cancer research in particular, the physiological consequences
that, using the GCM’s CO2 control function, long-term studies
of hypoxia are intensely investigated, as a low oxygen level is
for the investigation of biological processes in viable cells
a hallmark of most solid tumors (7).
become feasible (1).
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Using the Infinite 200 PRO with the GCM, it is now possible to
combine the induction of hypoxia inside the reader’s
measurement chamber with the subsequent measurement of
physiological parameters in cells or bacteria. This has the
following benefits that counteract the above mentioned
shortcomings:

No additional incubator is needed.

Transport from an incubator to a reader is unnecessary,
avoiding the need for additional manual work or
expensive robotic solutions.

No change of environmental conditions – which could
influence results – between hypoxia induction and
Figure 1 Tecan’s patent pending Gas Control Module (GCM) is compatible
with the Infinite 200 PRO series.
Inducing hypoxic states in 2-D and 3-D cell culture systems by
lowering the partial pressure of oxygen in the environment is
commonly performed in specialized cell culture incubators,
such as the Binder CB 53 (8), that are capable of
simultaneously regulating CO2 and O2 levels. The CO2 level is
set to 5-10 % to maintain the proper pH of the medium,
ensuring the growth and viability of the cells. Simultaneously,
the O2 level is set to a very low level (0.2 – 2 %) to efficiently
establish a hypoxic environment. However, there are some
drawbacks associated with this common approach:

Standard cell culture needs to be performed in a second,
separate CO2 incubator with normal oxygen partial
pressure (normoxia, ~21 %), which means that two
incubators are needed for the experiment.

The molecular analysis of hypoxia markers (e.g. the
expression and transcriptional activity of hypoxiainducible factors HIF-1 and HIF-2), or measurements of
cell viability requires the transfer of samples and
microplates to a detection system such as a multimode
microplate reader. Consequently, additional manpower or
expensive robotic solutions are needed to transfer the
microplates between the incubator and the reader.

The transfer of the microplate between the incubator and
the reader significantly changes the hypoxic environment
which might influence the outcome of the study.
subsequent assay and readout.
This application note describes the experimental validation of
the Infinite 200 PRO reader using three different analytical
approaches. For all experiments, the induction of hypoxia
inside the reader was compared to that inside a cell culture
incubator capable of simultaneously controlling CO2 and O2
levels (Binder CB 53 (8)).
The first experiment describes the hypoxic induction of HIF-1
transcriptional activity in the CellSensor™ HRE-bla ME-180
cell line by measuring HIF transcriptional activity (9). In the
second experiment, HCT 116 human colon carcinoma cells
were cultivated under hypoxic conditions and the protein
levels of the hypoxia marker proteins HIF-1α, as well as
vascular endothelial growth factor (VEGF) protein, were
subsequently quantified by immunoblotting (HIF1-α/VEGF
Western blot). Finally, tirapazamine (TPZ), a
chemotherapeutic drug that is cytotoxic only under hypoxic
conditions, was tested by measuring the cell viability of HCT
116 human colon carcinoma cells using the Life
Technologies™ alamarBlue® assay system (10).
Life Technologies CellSensor HRE-bla ME-180 cell line
Hypoxia-inducible factors (HIFs) are transcription factors that
are activated in response to decreasing oxygen levels in the
cellular environment. At present, several different HIF reporter
assay systems are commercially available that allow for the
quantitative determination of HIF transcriptional activity.
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The CellSensor HRE-bla ME-180 cell line contains a beta-
Originally identified in 1972 during a drug discovery program
lactamase reporter gene under the control of the hypoxia
screening for new herbicides, its clinical use was first
response element (HRE), stably integrated into the ME-180
described by Zeman et al in 1986 (11). While TPZ has shown
cervix carcinoma cells. This cell line is responsive to
only limited effectiveness in clinical trials (12), it has been
deferoxamine and cobalt chloride, and can be used to probe
used as a lead compound to develop a number of newer
the hypoxia signaling pathway. It can be adapted for high
compounds with improved anti-cancer properties (13).
throughput screening of agonist or antagonist compound
libraries. Candidate drugs can also be tested for dose
Materials and Methods
response against this cell line.
Instruments
The signal of the reporter system is based on GeneBLAzer®

Infinite F200 PRO filter-based multimode reader (Tecan,
substrate containing two fluoroprobes – coumarin and

Binder CB 53 CO2/O2 incubator (Binder GmbH, Germany)
fluorescein – which are both covalently attached to the

ChemiDoc™ MP imager (Bio-Rad Laboratories Inc., USA)
Austria)
technology. Cells are loaded with an engineered fluorescent
β-lactam ring of cephalosporin. In the absence of HIF
transcriptional activity, and hence no β-lactamase expression,
Software
the substrate molecule remains intact. In this state, excitation

i-control™ software (Tecan, Austria)
of the coumarin results in fluorescence resonance energy

Quantity One® 1-D Analysis software (Bio-Rad
transfer to the fluorescein moiety and emission of green light.
Laboratories Inc., USA)
However, in the presence of HIF activity, the expression of the
β-lactamase reporter enzyme is induced and the substrate is
Microplates
cleaved, separating the fluorophores and disrupting the

energy transfer. Consequently, excitation of coumarin in the
presence of the β-lactamase enzyme results in a blue
Thermo Scientific Nunc 96-well Edge plate, transparent,
cell culture treated (Thermo Fisher Scientific, USA)

fluorescent signal. The resulting blue/green ratio provides a
Thermo Scientific Nunc 6-well Multidish cell culture
treated (Thermo Fisher Scientific, USA)
normalized reporter response, quantitatively indicating the
extent of HIF activation in response to hypoxia.
Reagents and cell culture conditioning

alamarBlue is a proven cell viability indicator that uses the
CellSensor HRE-bla ME-180 cells (Life Technologies,
USA)
Life Technologies alamarBlue assay

DMEM (high-glucose); fetal bovine serum (FBS),
natural reducing power of living cells to convert resazurin to
dialyzed; non-essential amino acids (NEAA);
the fluorescent molecule resorufin. Resazurin is a redox
penicillin/streptomycin; HEPES (1 M, pH 7.3);
indicator that can be added directly to cells in culture. Viable
sodium pyruvate; Opti-MEM® reduced serum medium
cells convert the dark blue oxidized form of the dye (resazurin)
(Life Technologies, USA)

LiveBLAzer™ Loading Kit (CCF-4AM)
cells rapidly lose metabolic capacity and do not reduce

alamarBlue (Life Technologies, USA)
resazurin. Consequently, a fluorescent red signal is not

Mouse Anti-HIF-1α (BD Transduction Laboratories, USA)
generated.

Rabbit Anti-VEGF (Abcam, UK)

Rabbit Anti-Actin, (Sigma-Aldrich, Inc., USA)

Peroxidase-conjugated secondary antibodies (Dako,

ECL Plus™ Western blotting detection reagents
into a red fluorescent reduced form (resorufin; Ex 570 nm; Em
590 nm). This system is specific for cell viability, as non-viable
Tirapazamine (TPZ)
(Life Technologies, USA)
TPZ – 3-amino-1,2,4-benzotriazine-1,4 dioxide – is an
experimental anti-cancer drug that is transformed into a toxic
radical when the cells are exposed to very low levels of
Denmark)
(GE Healthcare Life Sciences, USA)
oxygen.
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
The CellSensor HRE-bla ME-180 cells were cultured in
HIF-1 protein detection via Western blotting
DMEM (10 % FBS dialyzed, 1 % penicillin/streptomycin,
HCT 116 cells were seeded into Thermo Scientific Nunc
0.1 nM NEAA, 1 mM sodium pyruvate and 25 mM
6-well cell culture plates at a density of 30,0000 cells/well and
HEPES. The assay was performed in Opti-MEM reduced
incubated overnight in a humidified atmosphere at 5 % CO2
serum medium with 0.5 % FBS dialyzed, 0.1 nM NEAA,
and 37 °C (Binder CB 53). To induce hypoxia, cells were
1 mM sodium pyruvate and 10 mM HEPES.
incubated at 37 °C and 5 % CO2 for five hours at normal
(21 %) or 0.2 % O2 concentrations in either a conventional cell
SDS PAGE
culture incubator (Binder CB 53) or the Infinite F200 PRO with

Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Pack
GCM. After five hours incubation in the hypoxic environment,
(Bio-Rad Laboratories Inc., USA)
cells were directly lyzed in Laemmli buffer. Proteins were

Trans-Blot Turbo Transfer System
separated by SDS PAGE, and transferred to a nitrocellulose
(Bio-Rad Laboratories Inc., USA)
membrane (Trans-Blot Turbo Mini Nitrocellulose Transfer
Pack) using the Trans-Blot Turbo Transfer System. Protein
Assay protocols
levels of HIF-1α, VEGF and actin were quantified using
specific antibodies. Chemiluminescence detection and signal
HIF reporter assay
quantification were then performed using ECL Plus Western
HRE-bla ME-180 cells were plated in the innovative 96-well
blotting detection reagents, the ChemiDoc MP imager and the
Nunc Edge assay plates (14) at a density of 20,000 cells/well
Quantity One 1-D Analysis software.
and incubated overnight at 5 % CO2 and 37 °C in a humidified
atmosphere. The Nunc Edge plate provides an evaporation
Cell viability after TPZ treatment
barrier enabling long-term incubation periods up to 75 hrs
HCT 116 cells were seeded into 96-well Nunc Edge plates at
inside the Infinite 200 PRO microplate reader without
a density of 1,000 cells/well and incubated overnight at 5 %
significant evaporation of the cell culture media (1). To induce
CO2 and 37 °C in a humidified atmosphere. TPZ was added in
hypoxia, cells were incubated at 37 °C and 5 % CO2 for eight
varying concentrations ranging between 0 and 100 µM,
hours, at normal (21 %), 1 % or 0.2 % O2 concentrations, in
followed by incubation in hypoxic conditions (5 % CO2, and 1
either a conventional cell culture incubator (Binder CB 53) or
or 0.2 % O2) for four hours in either the Binder CB 53
the Infinite F200 PRO with GCM. Cells were loaded with
incubator or the Infinite F200 PRO with GCM.
LiveBLAzer FRET B/G substrate and cultivated at room
temperature for a further two hours. Subsequently, the
After this treatment, the TPZ protocol required a 120 hrs
460/530 emission ratios were obtained using Infinite F200
incubation under normoxic conditions, which was performed
PRO settings optimized for GeneBLAzer-based assay
using a standard incubator. This very long incubation step can
systems (Table 1) and plotted for each condition (Figures 2-3).
also be performed inside the reader, to avoid manual transfer
of the plate. To minimize evaporation of the cell culture media,
For full automation, cells can be loaded with the LiveBLAzer
it is recommended refilling the evaporation moat of the Nunc
substrate using the reader’s injector system. The room
Edge plate every 48 hrs. However, it is important to take into
temperature incubation period can easily be performed inside
account that the reader will be blocked for 120 hrs; transfer of
the reader by using the software script to switch off the
the plate to a standard cell incubator might therefore be
heating after the addition of the substrate.
beneficial.
After incubation of the cells under normoxic conditions, the
alamarBlue cell viability assay was performed according to the
manufacturer’s instructions (10) using a six hrs incubation
period and the reader settings defined in Table 2.
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Again, no significant difference was observed between
Measurement settings
hypoxic induction and HIF transcriptional activity in the Binder
Measurement parameter
Donor
Acceptor
incubator and the Infinite F200 PRO with GCM.
emission
emission
Mode
FI bottom
FI bottom
Data shown in Figure 4 verify the results discussed above.
Plate
[NUN96.ft]
[NUN96.ft]
HIF-1α and VEGF protein levels were determined using
Excitation wavelength (nm)
415 (20)
415 (20)
standard immunoblotting procedures and showed similar
Emission wavelength (nm)
460 (20)
535 (25)
increases in both systems when the O2 level was adjusted to
0.2 %.
Flash number
25
25
Integration time
20 μs
20 µs
Lag time
0 sec
0 sec
Settle time
0 sec
0 sec
Mirror
automatic
automatic
Gain
optimal
optimal
Table 1 Infinite F200 PRO measurement settings for GeneBLAzer-based
reporter systems.
Measurement parameter
Instrument settings
Mode
FI top
Plate
[NUN96.ft]
Excitation wavelength
560 (10) nm
Emission wavelength
590 (20) nm
Flash number
25
Integration time
20 µs
Lag time
0 sec
Settle time
0 sec
Mirror
automatic
Gain
optimal
Figure 2 Relative activity (460/530 signal ratio) of HIF-1 transcription
factor under normoxic and hypoxic (1 % O2) conditions.
Table 2 Infinite F200 PRO measurement settings for the alamarBlue
assay (FI readout).
Results and discussion
Figure 3 Relative activity (460/530 signal ratio) of HIF-1 transcription
factor under normoxic and hypoxic (0.2 % O2) conditions.
Figures 2 and 3 represent the results obtained with the
CellSensor HRE-bla ME-180 cell line. At an O2 level of 1 %,
induction of hypoxia, and consequently HIF transcriptional
activity, was comparable between the Binder incubator and
the Infinite F200 PRO with GCM (Figure 2). The difference in
relative HIF-1 activities of the two systems is not significant,
ranging between four- to five-fold of normoxic control.
When using an O2 level of 0.2 %, hypoxia was induced even
more clearly, showing a 9- to 10-fold increase compared to
Figure 4 Expression levels of HIF-1 and VEGF under normoxic and
hypoxic (0.2 % O2) conditions. Relative expression levels were
calculated by normalization to actin expression (loading control).
normoxic control (Figure 3).
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The facultative chemotherapeutic TPZ was demonstrated to
Conclusion
be cytotoxic only under hypoxic conditions. Cytotoxic activity
was exhibited when HCT 116 colon cancer cells were
The results of the experiments described in this white paper
incubated in the presence of the drug for four hours under
clearly demonstrate the capability of the Infinite 200 PRO and
hypoxic conditions (1 % O2, Figures 5). The cytotoxic activity
the GCM to efficiently induce hypoxia. In all experimental
of TPZ was equivalent in the Binder incubator and the Infinite
approaches, the system was shown to be as effective as a
200 PRO multimode reader. In addition, the cytotoxic
well-established cell culture incubator that is widely used to
properties of TPZ were further increased by the incubation of
induce hypoxia in various cellular models. By maintaining a
HCT 116 cells at 0.2 % O2 for four hours (Figure 6).
high CO2 level (5 %) inside the reader’s measurement
chamber, and using the anti-evaporation Nunc Edge plate, cell
viability was maintained, while the reduction of the O2 level
induced hypoxia. The benefits of using a microplate reader to
induce hypoxia – rather than a standard incubator as
described in the introduction – makes the combination of the
Infinite 200 PRO reader with Tecan’s patent pending Gas
Control Module the perfect tool for all customers focusing on
hypoxia-related research.
Figure 5 Tirapazamine dose-response curve after hypoxia induction at
1 % O2. Data represent percent of untreated control.
Figure 6 Tirapazamine dose-response curve after hypoxia induction at
0.2 % O2. Data represent percent of untreated control.
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Abbreviations
8.
http://www.binder-world.com/us/products/co2-
9.
http://products.invitrogen.com/ivgn/product/K1644
incubators/cb-series/cb-53/
HIF
HRE
TPZ
VEGF
Hypoxia inducible factor
Hypoxia response element
Tirapazamine
Vascular endothelial growth factor
References
1.
Application Note. Analyzing biological processes.
Long-term cell-based kinetics using Tecan’s GCM and
the Thermo Scientific Nunc Edge plate. Tecan Application
Note 397050 V.1.0, 01-2012
2.
Sims N.R., Muyderman H. Mitochondria, oxidative
metabolism and cell death in stroke. Biochim Biophys
Acta 1802(1):80-91, 2010
3.
Vaupel P., Mayer A. Hypoxia in cancer: significance and
impact on clinical outcome. Cancer Metastasis Rev.
10. www.invitrogen.com
11. Zeman E.M, Brown J.M, Lemmon M.J., Hirst V.K., Lee
W.W. SR-4233: a new bioreductive agent with high
selective toxicity for hypoxic mammalian cells. Int J
Radiat Oncol Biol Phys 12(7), 1239-42, 1986
12. Reddy S.B., Williamson S.K. Tirapazamine: a novel agent
targeting hypoxic tumor cells. Expert Opin Investig Drugs
18(1), 77.87, 2009
13. Hay M.P., Hicks K.O., Pchalek K., Lee H.H., Blaser A.,
Pruijn F.B., Anderson R.F., Shinde S.S., Wilson W.R.,
Denny W.A. Tricyclic [1,2,4]triazine 1,4-dioxides as
hypoxia selective cytotoxins. J Med Chem 51(21),
6853-65, 2008
14. www.thermoscientific.com/edgeplate
26:225, 2007
4.
Brown J.M., Wilson W.R. Exploiting tumor hypoxia in
cancer treatment. Nat Rev Canc 4,437, 2004
5.
Boyle R.G., Travess S. Hypoxia: targeting the tumor.
Anticancer Agents Med Chem 64:281, 2006
6.
Cataldi A., Di Giulio C. ‘Oxygen supply’ as modulator of
aging processes: hypoxia and hyperoxia models for aging
studies. Cur Aging Sci 2(2):95-102, 2009
7.
Brown J.M., Giaccia A.J. The Unique Physiology of Solid
Tumors: opportunities (and Problems) for Cancer
Therapy. Cancer Research 58, 1408-1416; 1998
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397530 V1.0, 06-2012
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