Download Extracellular O2 Probe

ab140097,
ab140098 & ab140099 –
Extracellular O2 Probe
Instructions for Use
For the measurement of extracellular oxygen
consumption by isolated mitochondria, cell
populations, small organisms, tissues and
enzymes.
This product is for research use only and is not
intended for diagnostic use.
1
Table of Contents
1.
Introduction
3
2.
Product Overview
4
3.
Protocol for Cell-Based Screening of Mitochondrial Analysis
5
4.
Protocol for Plate Based Analysis of Isolated Mitochondria
11
5.
Assay Throughput and Performance
13
6.
Assessment of Classical Mitochondrial Effectors
15
7.
Compound Screening
17
2
1. Introduction
Mitochondrial dysfunction is implicated in numerous disease states
and is also a major mechanism of drug-induced toxicity. Oxygen
consumption is one of the most informative and direct measures of
mitochondrial function. Traditional methods of measuring oxygen
consumption are hampered by the limitations of low throughput and
high complexity. The Extracellular O2 probe assay from Abcam
solves these limitations by allowing convenient, plate-based analysis
of mitochondrial function. The assay is based on the ability of O₂ to
quench the excited state of the Extracellular O2 probe. As the test
material respires (e.g. isolated mitochondria, cell populations, small
organisms, tissues and enzymes), O₂ is depleted in the surrounding
solution/environment, which is seen as an increase in probe
phosphorescence signal. Changes in oxygen consumption reflecting
changes in mitochondrial activity are seen as changes in
Extracellular O2 probe signal over time.
The assay is non-chemical and reversible; a decrease in oxygen
consumption (an increase in O₂ levels) is seen as a decrease in
probe signal.
Extracellular O2 probe is analysed on standard fluorescence plate
readers
using
standard
96-well
microtitre
plates.
The
Extracellular O2 probe assay combines the high data quality and
information content of the oxygen electrode approach, with the
throughput and convenience of microtitre plate based assays. These
3
capabilities make Extracellular O2 probe the ideal tool for rapid
compound screening, IC50 generation and the application of
structure-activity relationship approaches.
2. Product Overview
The Extracellular O2 probe is supplied as a dry, stable and soluble
reagent that is easily reconstituted in ultra-pure water for simple
dispensing with a pipette. One vial of Extracellular O2 is used probe
per 96-well plate.
The Extracellular O2 probe for plate-based extracellular analysis is
provided in three different formats:
1. Extracellular O2 Probe (ab140097): comprises 1 vial of
Extracellular O2 probe only.
2. Extracellular O2 Probe (with mineral oil) (ab140098):
contains 1 vial of Extracellular O2 probe and 1 bottle of
mineral oil.
3. Extracellular
O2
Probe
(with
high-sensitivity
oil)
(ab140099): contains 1 vial of Extracellular O2 probe and 1
bottle of high-sensitivity oil.
Use
with
ab140100
Cellular
Acid
Extrusion
Probe
to
simultaneously measure oxygen consumption and pH changes on a
plate-reader.
For
the
intracellular
analysis
of
molecular
oxygen,
use
ab140101 Intracellular O2 Probe.
4
3. Protocol for Cell-Based Screening of
Mitochondrial Analysis
Assay Summary
Add Cells, Extracellular O2 Probe and Compound to 96-well plate
Add mineral oil to wells
Read plate using fluorescence plate reader
3.1. Plate Preparation
3.1.1. Equilibrate plate temperature on a plate heater to
30°C.
3.1.2. Add 150 µL of cells to test wells (suspension/
trypsinised adherent cells). Alternatively, adherent
cells may be pre-plated and 150 µL of fresh media
added prior to measurement.
3.1.3. Reconstitute Extracellular O2 Probe in 1 mL
ultra-pure H2O to give a 1 µM stock solution and
dispense 10 µL of this stock solution into to each
well.
3.1.4. For compound testing, add 1 µL of compound in an
appropriate solvent to test wells. If required,
incubate cells with compounds.
5
3.1.5. Add a 100 µL layer of mineral oil (preheated to
30°C) volume to each well. This increases assay
sensitivity by minimizing interference from ambient
oxygen.
3.2. Measurement
3.2.1. Insert the prepared plate into a fluorescence plate
reader pre-set to 30°C.
3.2.2. Measure Extracellular O2 Probe signal at 1.5 min
intervals for 30-200 min using excitation and
emission wavelengths of 380 nm and 650 nm
respectively.
(Readers
time-resolved
mode,
equipped
may
achieve
with
a
improved
performance using delay and gate time of 30 and
100 µsec).
3.2.3. Cell
respiration
causes
a
reduction
in
the
concentration of dissolved oxygen in the sample,
resulting in an increase in Extracellular O2 Probe
signal.
Note: Sufficient cell numbers are required to produce
measurable signal changes. The cell numbers are cell-type
dependent. Highly glycolytic adherent cells must be
trypsinised and concentrated prior to measurement.
6
3.3. Data Analysis
For semi-quantitative analysis, the rate of signal increase
can be used to compare treated and untreated samples or
to compile dose response data.
3.4. Monitoring Cell Respiration
The ability of the Extracellular O2 Probe assay to assess
cell respiration is illustrated in Fig. 2. Dilutions curves for
HepG2 cells (Fig. 1A) and primary rat hepatocytes (Fig. 1B)
are presented.
Fig. 1: Cell dilutions measured on 96-well plates using
Extracellular O2 Probe. A) HepG2 cell dilution after an
overnight (□) or 2 day culture period (■), B) primary rat
hepatocyte cell dilution after an overnight culture period.
Rates of probe signal change (slope of fluorescence signal)
were normalised against initial intensity.
7
3.5. Extracellular O2 probe and ATP analysis of cells
To contrast the sensitivities of oxygen consumption and
cellular ATP concentrations as indicators of mitochondrial
dysfunction; HepG2 cells were treated with a panel of
classical electron transport chain (ETC) modulators and
both cellular oxygen consumption and cellular ATP
concentrations were measured.
8
Fig 2: Parallel analysis of classical inhibitors on HepG2
cells. A) Extracellular O2 Probe assessment of mitochondrial
function immediately post treatment, B) analysis of ATP
concentrations 24 h post-treatment. For Extracellular O2
Probe measurements, cells were plated at 80,000 cells/well,
allowed to adhere overnight and then assayed. Extracellular
O2 data presented in Fig. 2A illustrates that drug induced
mitochondrial dysfunction is evident immediately post
treatment with both inhibition (Oligomycin, Rotenone,
Antimycin) and uncoupling (FCCP) being detected. Despite
this dysfunction and an additional 24 hour exposure,
analysis of cellular ATP concentrations indicated high levels
of ‘viability’ (Fig. 2B). This pattern is also reflected using
other assays and cell lines.
3.6. Extracellular O2 Probe: comparison with other methods
of cell screening
The dose-response relationship for the ETC inhibitor
rotenone using four different viability assays: Extracellular
O2 probe, Bioluminescent ATP Measurement, LDH and
Cellular DNA Measurement outlined in Fig. 3. These data
indicate
that
treatment
suppresses
mitochondrial
respiration, leading to loss of function and reduced oxygen
consumption prior to cell death.
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Fig. 3: Effects of Rotenone treatment on H-4-II-E cells
analysed using Extracellular O2 probe, Bioluminescent ATP
Measurement, LDH and Cellular DNA Measurement (24h
exposure).
These results demonstrate the effectiveness of Extracellular O2
Probe in assessing mitochondrial function in whole cells as well as
the importance of evaluating mitochondrial function in the context of
drug toxicity. The specificity and sensitivity of the Extracellular O2
Probe assay is demonstrated by its ability to detect mitochondrial
toxicity more rapidly and at lower drug doses than other assays.
When combined with other assays, Extracellular O2 Probe allows
detailed evaluation of mechanisms of drug toxicity, adding
significantly to the portfolio of information available for compound
evaluation.
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4. Protocol for Plate Based Analysis of
Isolated Mitochondria
Assay Summary
Add Cells, Extracellular O2 Probe +/- ADP and Compound to 96-well
plate
Add mineral oil to wells
Read plate using fluorescence plate reader
4.1. Plate Preparation
4.1.1.
Equilibrate plate temperature on a plate heater to
30°C.
4.1.2.
Reconstitute Extracellular O2 Probe in 1 ml H2O to
give a 1 µM stock solution. Dilute 1:10 in
measurement buffer (250 mM sucrose, 15 mM KCl,
1 mM EGTA, 5 mM MgCl2, 30 mM K2HPO4, pH 7.4)
and add 100 µl to each well.
4.1.3.
For compound testing, add 1 µL of compound in an
appropriate solvent to test wells.
4.1.4.
Dilute Mitochondria to the desired concentration in
measurement buffer (250 mM sucrose, 15 mM KCl,
1 mM EGTA, 5 mM MgCl2, 30 mM K2HPO4, pH 7.4)
and add 50 µL to test well. Dissolve substrate
(succinate
or
glutamate/malate)
and
ADP
in
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measurement buffer and add 50 µL of this solution
to test wells giving a final substrate concentration of
25
Mm
(succinate)
or
12.5/12.5
mM
(glutamate/malate) and a final ADP concentration of
1.65 mM.
4.1.5.
Add 100 µL of mineral oil (preheated to 30°C) to
each well. This increases assay sensitivity by
minimising interference from ambient oxygen.
Note: Plate preparation time should be kept to a
minimum.
4.2. Measurement
4.2.1. Insert the prepared plate into a fluorescence plate
reader preset to 30°C.
4.2.2. Measure probe signal at 1.5 min intervals for
10-30
min
using
excitation
and
emission
wavelengths of 380 nm and 650 nm respectively.
(Readers equipped with a time-resolved mode, may
achieve improved performance using delay and gate
time of 30 and 100 µsec respectively).
Note: Mitochondria are freshly prepared as per user’s
protocol and should not be left on ice for longer than
recommended in the literature. Measurement buffers should
be prepared freshly on the day of measurement.
12
4.3. Data Analysis
• For semi-quantitative analysis the rate of signal increase
can be used to compare treated and untreated samples
or to compile dose response data.
• For more quantitative analysis, fluorescence data may
be related to oxygen concentrations.
5. Assay Throughput and Performance
The data output from the Extracellular O2 Probe assay (96 samples),
and polarographic (one sample) analysis of mitochondrial oxygen
consumption is contrasted in Fig. 4. Fig 4a shows typical
polarographic analysis illustrating initiation of State 2 and State 3
respiration through addition of substrate and ADP respectively. Fig
1b shows oxygen consumption measured using Extracellular O2
Probe, with glutamate/malate- (left) and succinate (right) -driven
oxygen consumption is measured at decreasing mitochondrial
protein concentrations in both State 2 (top) and State 3 (bottom)
(n = 4).
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Fig. 4: Analysis of isolated mitochondrial using
A)
conventional polarography and B) Extracellular O2 Probe. C)
Data extracted from Fig. 4B illustrating activation of
succinate
driven
mitochondrial
measured
using
Extracellular
oxygen
O2
consumption
Probe
A65N
(n = 4, % CV < 3 %).
14
The compatibility of the Extracellular O2 Probe assay with the
microplate format permits analysis under 96 (or 384) discrete
conditions. The effectiveness of this level of throughput in
analysing isolated mitochondria is highlighted in Fig. 2B which
examines increasing mitochondrial protein concentrations on
glutamate/malate- and succinate-driven respiration in both basal
(State
2)
and
ADPactivated
(State
3)
states
(all
in
quadruplicates).
The performance of the Extracellular O2 Probe assay is
highlighted in Fig. 4C, with coefficient of variance below 3%.
6. Assessment of Classical Mitochondrial
Effectors
Validation of Extracellular O2 Probe for assessment of
mitochondria is illustrated in Fig. 5. These data illustrate the
inhibition of mitochondrial function using a panel of classical
mitochondrial inhibitors and highlight the dose dependence of
this inhibition for KCN.
15
Fig. 5: A) Monitoring the effect of a panel of classical ETC
inhibitors on mitochondrial function using Extracellular O2
probe, and B) dose dependent inhibition of mitochondrial
function by KCN.
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7. Compound Screening
Extracellular O2 Probe allows screening of compounds at
multiple concentrations and in multiple conditions in a single
microtitre plate as illustrated in Fig. 6. Such data may be
processed further to generate dose response data.
Fig. 6: Effect of test compounds (D01-D05), on both State 2
and
State
3
isolated
mitochondrial
function
using
Extracellular O2 probe. Some compounds uncouple in a
dose dependant manner while others inhibit.
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Overall, Extracellular O2 Probe allows highly sensitive highthroughput detection of mitochondrial dysfunction in isolated
mitochondria. Use of a 96-well plate format allows screening of
200 compounds per day at a single dose, or acquisition of dose
response characteristics for 25 compounds per day. This
capability represents a fundamental shift in the capacity for
mitochondrial toxicity testing in drug discovery programs, without
compromising data quality or information content.
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