Real-time Monitoring of Intracellular
Oxygen Using MitoXpress®-Intra
James Hynes and Conn Carey , Luxcel Biosciences Ltd, Ireland
Catherine Wark, BMG LABTECH, Ltd, UK
Application Note 223, Rev. 03/2012
Convenient, real-time monitoring of intracellular oxygen within
a cell monolayer using the FLUOstar Omega.
Membrane permeable, ultra-sensitive, probe increases as
oxygen levels are depleted within the cell.
Easily assess transient changes in metabolic activity, as well as
hypoxia, which extracellular probes cannot do.
Introduction
MitoXpress®-Intra is an oxygen-sensitive probe developed for
the intracellular analysis of molecular oxygen using plate-based
time-resolved fluorometry. Measurement is based on the ability
of O2 to quench the emission of a probe which is taken up by
endocytosis. As cells respire, the concentration of oxygen within
the cell monolayer is depleted and this depletion is seen as
an increase in probe signal, expressed as probe lifetime, following
ratiometric measurement of time-resolved phosphorescence
intensity. This allows real-time information on the concentration
of molecular oxygen within the cell monolayer to be generated
across multiple samples without a requirement for specialised
imaging equipment. The ability to monitor such fluctuations is
particularly informative as it allows the assessment of transient
changes in metabolic activity and provides access to a critical
parameter in the study of hypoxia which is beyond the capacity of
extracellular sensing methodologies.
Materials and Methods
MitoXpress®-Intra (NanO2) reagent from Luxcel Biosciences
FLUOstar Omega microplate reader equipped with Atmospheric
Control Unit (ACU) (Fig. 1)
Probe preparation and cell loading
Cells are grown to full confluence in a suitable medium
supplemented with foetal bovine serum, L-glutamine and Penicillin/
Streptomycin on cell culture treated 96 well plates. Replace seeding medium with 100 µl of normal growth media
(10%FBS) containing MitoXpress®-Intra probe at 10µg/ml and
return to culture overnight. Prior to measurement, replace media with
150 µl of pre-warmed media.
Measurement
Intracellular oxygen in samples is measured using the BMG
LABTECH FLUOstar Omega and the scripting function. Optimal filter
wavelengths are 340 TR L for excitation and EM 655-50 for emission.
Phosphorescent intensities are measured at delay times of 30 μs
and 70 μs (with 30 μs window time) with the ratio of these
intensities subsequently converted to phosphorescent lifetimes as
shown in Figure 2. These conversions are performed with the help of
the MARS data anaylsis software.
Intensity
Assay Principle
Fig. 1: BMG LABTECH’s FLUOstar Omega multidetection microplate reader
with the Atmospheric Control Unit (ACU).
Intensity
Molecular oxygen is a key substrate of all aerobic organisms and the
terminal acceptor of the electron transport chain (ETC). This highly
informative marker of cellular metabolism and mitochondrial function
is closely regulated by the ATP/ADP ratio, the levels of available oxygen
and the action of signalling molecules such as NO and Ca2+.
Analysis of molecular oxygen facilitates the elucidation of
critical biochemical pathways; including cell survival and death,
mitochondrial function, toxicological impact of compounds and
metabolic alterations caused by various stimuli or disease states.
To date, routine analysis of intracellular molecular oxygen has
been limited by the lack of suitable means of sensing oxygen
within the cell monolayer.
30
µsec
t1
70
µsec
Time
t2
Time
Fig. 2: Measurement principle for calculation of phosphorescence lifetime
from ratiometric time-resolved phosphorescence intensity.
Results and Discussion
Typical Data Output
When analysing a fully confluent monolayer, ETC activity results in
measurement usually beginning at oxygen concentrations below
air saturated levels. Reduced ETC activity results in an increase in
intracellular oxygen concentration resulting in a decrease in probe
signal while increased ETC activity results in a decrease in intracellular
oxygen concentration resulting in an increase in probe signal (Fig 3).
Increases may be prolonged or more transient, depending on the
processes involved and the measurement conditions.
Activation
Assay Performance & Cell Responses
Figure 5 shows the effect of HepG2 cells treated with an electron
transport chain inhibitor (Antimycin) and uncoupler (FCCP). The
magnitude, consistency of monolayer (de)oxygenation is monitored.
Profiles are seen to be highly consistent with uncoupling causing
enhanced oxygen consumption seen by an immediate decrease in
intracellular oxygen resulting in an increase in probe lifetime while
inhibition causes the reverse. Increasing the FCCP concentration
increases the speed of the deoxygenation while increasing the cell
number increases the depth of the deoxygenation (data not shown).
Treatment of HCT116 cells with the RyR1 agonist ryaniodine also
causes a transient deoxygenation of the monolayer via a calcium
mediated increase in metabolic rate (Fig. 5B), a response not readily
apparent using extracellular analysis.
A
0%
56
Treatment
FCCP
Lifetime (µs)
Fluorescent Signal
52
Resting
48
44
40
36
DMSO
32
Antimycin
28
O2
Inhibition
O2
24
4 min 47 s
10 15 min 11 s
20
24 min 48 s
30
35 min 12 s
40
44 min 47 s
50
55 min 12 s
60
21%
Time Post Treatment (min)
Time
Fig. 3: Diagrammatic representation of MitoXpress®-Intra probe profiles in
response to drug treatment.
B
50
Growth Media Addition
2 µM Ryanodine
Probe Response
Measurement of phosphorescent lifetime of the MitoXpress®-Intra
probe facilitates measurement of the basal level of intracellular
oxygen within the cell monolayer. The response of the probe is
demonstrated in Antimycin treated HepG2 cells measured on
a FLUOstar Omega equipped with an atmospheric control unit
(BMG LABTECH UK). This instrument allows the ambient O2
concentration to be reduced stepwise. The time taken for the
non-respiring monolayer to equilibrate to this new O2 concentration
can be observed (Fig. 4) with the new steady state probe signal
reflecting this new concentration. Such calibrations also allow
measured lifetime values to be automatically transposed into O2
concentrations, thereby allowing probe response to be viewed in
this scale.
0%
46
Lifetime [µs]
46
Treatment
42
38
34
30
0
10
20
30
40
50
Time [min]
Fig. 5: Consistency of response & inhibition and uncoupling of ETC activity (A)
and kinetic analysis of metabolic responses agonist treatment (B).
Conclusion
MitoXpress®-Intra allows the convenient monitoring of intracellular
oxygen on a conventional microtitre plate format. The probe is ‘self
loading’ and provides excellent signal to blank performance. This
allows the investigation of a variety of parameters currently beyond
the capabilities of existing probe technologies.
Lifetime (µs)
42
38
34
30
26
O2
22
20
40
60
80
100
120
140
160
21%
Time(min)
Fig. 4: Kinetic probe trace showing the response of the cell monolayer to
decreasing ambient oxygen.
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