Hynes 2016

Fluorescence-Based Microplate Assays UNIT 2.
16
for In Vitro Assessment of Mitochondrial
Toxicity, Metabolic Perturbation,
and Cellular Oxygenation
James Hynes,1 Conn Carey,1 and Yvonne Will2
1
Luxcel Biosciences, BioInnovation Centre, University College Cork, Cork, Ireland
2
Pfizer Global Research, Groton, Connecticut
High-throughput in vitro cell metabolism assays are of particular use for iden-
tification and delineation of mitochondrial toxicity and related metabolic per-
turbation. Here, a panel of fluorescence-based metabolism assays are described
for measuring oxygen consumption, glycolytic flux, and cellular oxygenation.
They can be applied to analysis of both isolated mitochondria and cell mod-
els. Sample data are presented illustrating how these protocols can be used to
examine drug treatment, the interplay between oxidative and glycolytic ATP
generation, and the impact of cell oxygenation on this balance. Descriptions are
provided on how these measurements can be applied to 3D systems and how
they can be multiplexed with other relevant metabolic readouts. Mitochondrial
isolation and cell permeabilization protocols are also provided.
C 2016 by John
Wiley & Sons, Inc.

Keywords: 3D culture r cellular oxygenation r glycolysis r mitochondria r
oxygen consumption r toxicity
How to cite this article:

Hynes, J., Carey, C., and Will, Y. 2016. Fluorescence-based
microplate assays for in vitro assessment of mitochondrial toxicity,
metabolic perturbation, and cellular oxygenation. Curr. Protoc.
Toxicol. 70:2.16.1-2.16.30.
doi: 10.1002/cptx.3
INTRODUCTION
Recent years have seen a growing appreciation for the importance of the mitochondrion
as a site for off-target effects of drug therapy (Wallace, 2008), which has led to major
organ toxicities (UNIT 2.15; Nadanaciva and Will, 2009) and contributed to compound
attrition and post-market drug withdrawals across a variety of drug classes. This is unsur-
prising when one considers both the multitude of sites where mitochondrial function can
be perturbed and the deleterious consequences of such perturbation. High-throughput-
screening-compatible in vitro methods for assessing metabolic implications of drug treat-
ment and identifying mechanisms by which such toxicities evolve are therefore of partic-
ular value. An important additional consideration is the biological relevance of the in vitro
model used, particularly as standard glucose-grown cell lines have the capacity to ame-
liorate the cytotoxic implications of mitochondrial insult (Marroquin et al., 2007). Mito-
chondria isolated from relevant tissue can be used for a specific and detailed assessment of
drug treatment on the isolated organelle, or testing can be performed in whole cells using
2D or 3D cultures, typically using liver or cardiac models (Marroquin et al., 2007; Hynes
et al., 2013). Additionally, focus is also now shifting to the culture and testing conditions
used, with a growing desire to perform such analysis under more physiologically relevant Assessment of Cell
Toxicity
Current Protocols in Toxicology 2.16.1-2.16.30, November 2016 2.16.1

Published online November 2016 in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cptx.3 Supplement 70
Copyright C 2016 John Wiley & Sons, Inc.
conditions by providing relevant respiratory substrates and biologically relevant oxygen
concentration, as opposed to the supraphysiological glucose and oxygen concentrations
often used (Chapple et al., 2016).
Oxygen consumption measurements are arguably the most informative indicator of mito-
chondrial function (Hynes et al., 2006), as they allow a direct and specific assessment of
the functioning of the electron transport chain (ETC), the cornerstone of oxidative phos-
phorylation and cellular metabolism. Additional information on glycolytic metabolism
can be obtained by assessing extracellular acidification, thereby providing information
on the two main sources of cellular ATP. A key additional parameter is the level of
oxygen available to the in vitro model, as this can impact both metabolic poise and
metabolic response to insult. The following protocols and related commentary outline
how O2 - and pH-sensitive fluorophores can be used as tools for convenient microplate-
based assessment of mitochondrial function and metabolic perturbation. Four methods
are described: (1) measurement of oxygen consumption in isolated mitochondria (Basic
Protocol 1); (2) measurement oxygen consumption in 2D and 3D cell models (Basic
Protocol 2); (3) measurement of extracellular acidification for assessing glycolytic flux
in 2D and 3D cell models, including multiplexing with O2 measurements (Basic Protocol
3); and (4) measurement of cellular oxygenation in 2D and 3D cell models, including
multiplexing with glycolytic flux analysis (Basic Protocol 4). Sample data are presented
to illustrate how these protocols can be used to examine changes in the interplay be-
tween oxidative and glycolytic ATP generation, and the impact of cellular oxygenation
on this interplay, thereby facilitating the interrogation of metabolic responses to drug
treatment and other metabolic phenomena such as the Warburg effect. Support protocols
provide detailed information on isolation of mitochondria (Support Protocol 1); mea-
surement of mitochondrial protein concentration (Support Protocol 2); optimization of
plate reader settings (Support Protocol 3); cell permeabilization for mechanistic analysis
(Support Protocol 4); and calibration of intracellular oxygen measurements (Support
Protocol 5).
MEASUREMENT OF OXYGEN CONSUMPTION IN ISOLATED

MITOCHONDRIA
The impact of a drug on mitochondrial function can be assessed specifically by measur-
ing its effect on oxygen consumption in isolated mitochondria (Hynes et al., 2006). The
oxygen consumption rate produced by a given concentration of mitochondrial protein is
dependent on the substrate provided and the availability of ADP (Hynes et al., 2006).
Therefore, it is necessary to establish the optimum protein concentration for analysis
of both basal and ADP-driven respiration before examining drugs for potential mito-
chondrial effects. Basic Protocol 1 outlines how to determine the optimum mitochondrial
protein concentration to use for glutamate/malate and succinate-driven respiration in both
basal and ADP-stimulated conditions (Hynes et al., 2006, 2013; Chapple et al., 2016),
and describes the recommended procedure for screening compounds for mitochondrial
liabilities.
BASIC Measurement of Electron Transport Chain Activity

PROTOCOL 1
ETC, ATPase, and adenine-nucleotide translocator (ANT) inhibitors cause a decrease in
ADP-activated respiration, while uncouplers cause an increase in basal respiration (Hynes
et al., 2006). Traditionally, in vitro analysis of oxygen consumption in both mitochondria
and whole cells was conducted using a Clark electrode, but this approach lacks the
Fluorescent Assay
throughput required for large compound libraries and IC50 determination. Here, the use
for Mitochondrial of an oxygen-sensitive fluorescent probe (MitoXpress-Xtra) facilitates high-throughput
Function microplate-based analysis of ETC activity in isolated mitochondria.
2.16.2
Supplement 70 Current Protocols in Toxicology
Materials
MitoXpress-Xtra HS Oxygen Consumption Assay kit (Luxcel Biosciences, cat. no.
MX-200), including high-sensitivity (HS) mineral oil
Measurement buffer 1 (see recipe)
Substrate stock solution (select one; store in aliquots at −20°C):
1 M sodium succinate in water, pH 7.4
0.5 M glutamate/0.5 M malate in water, pH 7.4
100 mM adenosine 5 -diphosphate (ADP) in water (store in aliquots at −20°C)
Isolated mitochondria (see Support Protocol 1)
Test compounds in appropriate solvent
Positive control compounds: rotenone and p-trifluoromethoxy carbonyl cyanide
phenylhydrazone (FCCP)
Fluorescence plate reader (Table 2.16.1) with temperature control and kinetic
analysis software
30°C water bath
96-well plates, black with clear bottom
Multiblock plate heater (VWR, cat. no. 12621-108)
Multichannel pipetter
Syringe dispenser (Eppendorf) with 2.5-ml plastic syringes
Prepare instrument and reagents
1. Warm the plate reader to 30°C and prepare the kinetic measurement protocol with
appropriate settings to read the desired wells at 0.5- to 1.5-min intervals over 20 to
30 min.
Prior to use, ensure that instrument settings have been optimized (Table 2.16.1 and
Support Protocol 3). Three discrete fluorescence modalities can be used, depending on
the plate reader: intensity measurement (basic), time-resolved fluorescence (TR-F) mea-
surement (standard), and dual-read ratiometric TR-F measurement (lifetime calculation)
(advanced). See Background Information for additional discussion.
2. Reconstitute MitoXpress-Xtra probe in 1 ml measurement buffer 1, then dilute to

10.5 ml with the same buffer.
The standard probe package is designed for one 96-well plate. Once reconstituted, the
undiluted probe can be stored in the dark for several days at 4°C. Diluted probe should
be used on the same day.
3. Warm mineral oil and diluted probe to 30°C.

Set up assay plate
NOTE: To minimize oxygen depletion in samples prior to measurement, plate preparation
time should be kept to a minimum.
For optimization of mitochondrial protein concentration

4a. Prepare substrate solution as follows and warm to 30°C:
For basal respiration (State 2):

150 µl substrate stock solution
1.35 ml measurement buffer 1.
For ADP-stimulated respiration (State 3):
100 µl of 100 mM ADP
1.25 ml measurement buffer 1. Assessment of Cell
Toxicity
2.16.3
Current Protocols in Toxicology Supplement 70
Table 2.16.1 Recommended Settings for Common Fluorescence Microplate Readersa
Excitation/emission
Manufacturer Instrument Optical configuration Readsb Optimum mode wavelengths (nm)
For MitoXpress measurements:
BMG FLUOstar Filter-based 30/30 µsec Dual-read TR-F 340 ± 50 nm (TR-EX L)
Labtech Omega/ Top or bottom read 70/30 µsec (lifetime) 655 ± 50 nm (BP-655)
POLARstar
Omega
CLARIOstar Filter-based 30/30 µsec Dual-read TR-F 340 ± 50 nm (TR-EX)
Bottom read 70/30 µsec (lifetime) 665 ± 50 nm or 645 ±
20 nm with LP-TR
dichroic
PHERAstar FS Filter-based 40/100 µsec TR-F 337 nm (HTRF module)
Top or bottom read N/A 665 nm (HTRF Module)
FLUOstar Filter-based 30/100 µsec TR-F 340 ± 50 nm(TR-EX L)
Optima/ Top or bottom read N/A 655 ± 50 nm (BP-655)
POLARstar
Optima
Perkin VICTOR Filter-based 30/30 µsec Dual-read TR-F 340 ± 40 nm (D340)
Elmer X4/X5 Top read 70/30 µsec (lifetime) 642 ± 10 nm (D642)
EnVision Filter-based 40/100 µsec TR-F 340 nm ± 60 nm (X340)
Top read N/A 650 nm ± 8 nm (M650)
BioTek Cytation 3/5 Filter-based 30/30 µsec Dual-read TR-F 380 ± 20 nm
Top or bottom read 70/30 µsec (lifetime) 645 ± 15 nm
Synergy Filter-based 30/30 µsec Dual-read TR-F 380 ± 20 nm
H1/Neo2 Top or bottom read 70/30 µsec (lifetime) 645 ± 15 nm
Tecan Spark 10M Filter-based 30/30 µsec Dual-read TR-F 380 ± 20 nm
Top or bottom read 70/30 µsec (lifetime) 650 ± 20 or 670 ±
40 nm
Infinite M1000 Monochromator/filter 30/30 µsec Dual-read TR-F 380 ± 20 nm
Pro/ F200 Pro Top or bottom read 70/30 µsec (lifetime) 650 ± 20 or 670 ±
40 nm
Infinite M200 Monochromator/filter 30/100 µsec TR-F 380 ± 20 nm
Pro/ Safire/ Top or bottom read N/A 650 ± 20 nm
Genios Pro
Molecular Gemini/ Monochromator N/A Intensity 380 nm
Devices Flexstation Top or bottom read N/A (Prompt) 650 nm
continued
5a. Prepare a six-point dilution series of isolated mitochondria in measurement buffer 1

with a 1.5-ml total volume for each concentration.
Recommended concentrations are 1.0, 0.5, 0.25, 0.125, 0.63, and 0.03 mg/ml mitochon-
drial protein.
6a. Place a black 96-well plate on a plate heater equilibrated to 30°C.

7a. Using an automatic or multichannel pipetter, dispense solutions according to the
plate map in Figure 2.16.1A:
Fluorescent Assay 100 µl diluted MitoXpress-Xtra probe

for Mitochondrial 50 µl mitochondria (at indicated dilutions)
Function
50 µl substrate solution.
2.16.4
Table 2.16.1 Recommended Settings for Common Fluorescence Microplate Readersa , continued
Excitation/emission
Manufacturer Instrument Optical configuration Readsb Optimum mode wavelengths (nm)
For pH-Xtra measurements:
BMG FLUOstar Filter-based 100/30 µsec Dual-read TR-F 340 ± 50 nm (TR-EX L)
Labtech Omega/ Top or bottom read 300/30 µsec (lifetime) 615 ± 10 nm (BP-615)
POLARstar
Omega
CLARIOstar Filter-based 100/30 µsec Dual-read TR-F 340 ± 50 nm (TR-EX L)
Top or bottom read 300/30 µsec (lifetime) 615 ± 10 nm (BP-615)
PHERAstar FS Filter-based 100/30 µsec Dual-read TR-F 337 nm
Top read (HTRF 300/30 µsec (lifetime) 620 nm
module)
FLUOstar Filter-based TR-F 340 ± 50 nm(TR-EX L)
Optima/ Top or bottom read 100/100 µsec 615 ± 10 nm (BP-615)
POLARstar N/A
Optima
Perkin VICTOR Filter-based 100/30 µsec Dual-read TR-F 340 ± 40 nm (D340)
Elmer X4/X5 Top read 300/30 µsec (lifetime) 615 ± 8.5 nm (D615)
EnVision Filter-based 100/50 µsec Dual-read TR-F 340 nm ± 60 nm (X340)
Top read 300/50 µsec (lifetime) 615 nm ± 8.5 nm
(M615)
BioTek Cytation 3/5 Filter-based 100/30 µsec Dual-read TR-F 360 ± 40 nm
Top or bottom read 300/30 µsec (lifetime) 620 ± 10 nm
Synergy Filter-based 100/30 µsec Dual-read TR-F 360 ± 40 nm
H1/Neo2 Top or bottom read 300/30 µsec (lifetime) 620 ± 10 nm
Tecan Spark 10M Monochromator- 100/30 µsec Dual-read TR-F 380 ± 20 nm
based 300/30 µsec (lifetime) 615 ± 10 nm
Top or bottom read
Infinite M1000 Monochromator/filter 100/30 µsec Dual-read TR-F 380 ± 20 nm
Pro/ F200 Pro Top or bottom read 300/30 µsec (lifetime) 615 ± 10 nm
Infinite M200 Monochromator/filter TR-F 380 ± 20 nm
Pro/ Safire/ Top or bottom read 100/100 µsec 615 ± 10 nm
Genios Pro N/A
a Where available, filter-based dual-read (lifetime) TR-F is recommended, particularly for pH-Xtra and MitoXpress-Intra measurements. It is required
for quantitative analyses, including data transposition into H+ and O2 scales.
b The upper number represents Read 1 (D1/W1) and the lower number represents Read 2 (D2/W2).
The final substrate concentration is 25 mM succinate or 12.5/12.5 mM glutamate/malate.

For State 3 analysis, the final ADP concentration is 1.65 mM.
8a. Using a syringe dispenser, quickly add 100 µl prewarmed mineral oil to each well,
taking care to avoid air bubbles. Proceed to measurement and analysis.
For compound screening
4b. Aliquot stock solutions of test compounds in a 96-well plate according to the plate
maps in Figure 2.16.1B,C. Dilute compounds with measurement buffer 1 to produce
known concentrations as follows:
For initial screening (yes/no effect): Assay at a single concentration (e.g., 100 nmol/mg) in Assessment of Cell
Toxicity
duplicate (left and right in Fig. 2.16.1B).
2.16.5
Figure 2.16.1 Recommended plate maps for analysis of oxygen consumption in isolated mi-
tochondria. (A) Setup for assay optimization. (B) Setup for single-concentration drug screening.
Here, compounds 1-46 are assayed in duplicate. (C) Set up for dose-response analysis. Here,
compounds 1-11 are assayed at two-fold serial dilutions.
For analysis of hits and generation of IC50 values: Prepare serial 1:2 dilutions for each
compound at one data point per individual concentration (Fig. 2.16.1C).
To reduce the volumes required, a 96-well PCR plate can be used.
Fluorescent Assay As drug concentrations are usually expressed in nmol/mg mitochondrial protein, different
for Mitochondrial dilutions need to be prepared depending on the chosen mitochondrial test concentration.
Function
The final DMSO content in the assay wells should not exceed 0.5% (v/v).
2.16.6
Rotenone serves as a positive control for inhibition, and FCCP serves as a positive
control for uncoupling. As FCCP exhibits a bell-shaped dose response, a concentration
should be chosen that provides maximal oxygen consumption. As a negative control, use
measurement buffer 1 with DMSO but no drug.
5b. Prepare substrate solution as follows and warm to 30°C:
For State 2 respiration (uncoupler screening):
For State 3 respiration (inhibitor screening):
400 µl of 100 mM ADP
6b. Prepare 6 ml mitochondria solution at the optimal working dilution (previously
determined using steps 4a-8a).
7b. Place a black 96-well plate on a plate heater equilibrated to 30°C.
8b. Using an automatic or multichannel pipetter, dispense solutions according to the
plate maps in Figure 2.16.1B,C:
100 µl diluted probe
1 µl compound at desired concentration
50 µl optimized mitochondria dilution
50 µl substrate solution.
9b. Using a syringe dispenser, quickly add 100 µl prewarmed mineral oil to each well,
taking care to avoid air bubbles. Proceed to measurement and analysis.
Perform measurement and analysis
10. Insert the microplate into the prewarmed plate reader and commence reading.
11. When the measurement cycle is complete, remove plate from the instrument and
save data to file.
12. Analyze fluorescence intensity data in raw form (basic analysis) or transpose it into
an oxygen scale using appropriate oxygen calibration (advanced analysis).
Basic analysis is the most common approach taken. Use the instrument’s data analysis
software to plot time profiles for each well. If such software is unavailable, export data to
a suitable data analysis package. Select the linear portion of the signal profile (avoiding
any initial lag or subsequent plateau) and apply linear regression to determine the slope
for each well. Tabulate the slope values for each test sample, calculating appropriate
average and standard deviation values across replicate wells.
Advanced analysis is best performed using ratiometric TR-F data. Oxygen conversion is
possible on some instrument analysis packages. If unavailable, lifetime can be calculated
manually using blank-corrected intensity values as follows: 40/ln(R1/R2), where R1 and
R2 represent read one and read two. This provides lifetime values with units of μsec.
These lifetime profiles can be converted to an oxygen scale using a default calibration
function: O2 % = 522.8 × (− τ /9.064). Data linearization can also be performed, if
required (Ogurtsov et al., 2008).
13. When optimizing the mitochondrial protein concentration:
a. Select as optimal the concentration that allows analysis of both inhibition and
uncoupling.
Assessment of Cell
Toxicity
2.16.7
Figure 2.16.2 Results of assay optimization and drug screening. (A) Sample assay readout measured as indicated in
Figure 2.16.1A. (B) Intensity respiration profiles for State 3 succinate-driven respiration (n = 4) showing that the rate of
probe signal change increases as protein concentration increases. (C) Succinate driven-respiration from 0.125 mg/ml
mitochondrial protein measured in the presence (State 3) and absence (State 2) of ADP. (D) Sample data output for
compounds 1-46 screened at a single dose using isolated rat liver mitochondria in state 3 as shown in Figure 2.16.1B.
(E) Processed data illustrating replicate measurements (solid and empty bars) of compounds 1-46. Inhibition is rep-
resented by a negative value; complete inhibition = −100%. For this analysis, samples showing >25% change are
considered responders.
Screens are typically performed at 0.5 mg/ml succinate or 1 mg/ml glutamate/malate

for basal respiration, or at 0.25 mg/ml succinate or 0.25 mg/ml glutamate/malate for
ADP-driven respiration.
b. Compare samples with ADP (State 3) and without ADP (State 2) for each in-
dividual substrate to obtain the State 3/State 2 ratio. See Figure 2.16.2 for raw
sample data.
The ratio of State 3/State 2—the respiratory control ratio (RCR)—is a measure of coupling
and is an important indicator of the quality of the mitochondrial preparation. The RCR
should be calculated using slopes derived from data in the oxygen scale or linearized data.
If a significant baseline drift is seen on the original profiles (i.e., at zero protein con-
centration), it is recommended that the baseline be subtracted from all profiles prior to
transformation. This usually improves the linearity of transformed plots.
14. To generate a dose-response curve, plot the generated data against the corresponding
compound concentration. For inhibitors, apply sigmoidal fits to this dependence or
plot it to determine IC50 values (for inhibitors) or UC50 values (for uncouplers).
15. Group compounds based on their effect on mitochondria and rank order based on
UC50 and/or IC50 values.
See Figure 2.16.2 for sample results.
SUPPORT Isolation of Mitochondria from Rat Liver

PROTOCOL 1
Fluorescent Assay This protocol is based on the method of Lapidus and Sokolove (1993). Protocols for
for Mitochondrial isolation of mitochondria from cardiac and skeletal muscle are available (Hynes et al.,
Function
2006; Will et al., 2007).
2.16.8
For best results, rats should be young and weigh between 150 and 180 g. They should be
housed in pairs in a controlled environment with constant temperature (21° ± 2°C) and a
12-hr light/dark cycle with food and water provided ad libidum. Additional information
can be found in the Guide for Care and Use of Laboratory Animals (NRC, 2011).
NOTE: All protocols using live animals must first be reviewed and approved by an Insti-
tutional Animal Care and Use Committee (IACUC) and must follow officially approved
procedures for the care and use of laboratory animals.
Materials
Male Sprague-Dawley rats (150 to 180 g; e.g., Charles River)
Carbon dioxide source
Isolation buffers 1 and 2 (see recipes), ice cold
Sterile dissection tools

100-ml glass tissue homogenizer with Teflon pestle
Power drill (hand-held or static)
Cheesecloth (VWR cat. no. 21910-105)
Glass stirring rods
Homogenize liver
1. Euthanize animals with an overdose of carbon dioxide.
Anesthetics should be avoided, as they can have adverse effects on mitochondrial quality.
2. Excise organs rapidly and place them into ice-cold isolation buffer 1 (10 ml/g liver).
3. Mince 5 to 7 g liver very finely with scissors and wash several times in 20 ml
isolation buffer 1 until the homogenate is free of blood.
4. Homogenize the tissue in 5 vol isolation buffer 1 using a smooth glass grinder with
a Teflon pestle driven by a power drill on low speed (six to eight passes).
5. Bring homogenate to 8 vol with isolation buffer 1.
Isolate mitochondria
6. Centrifuge homogenate 10 min at 700 × g, 4°C, to remove debris.
7. Filter through two layers of cheesecloth.
8. Centrifuge filtrate 10 min at 14,000 × g, 4°C, to precipitate the mitochondrial
fraction. Discard supernatant.
9. Wash mitochondrial pellet by resuspending in 20 ml isolation buffer 1 using a glass
stirring rod and centrifuging 10 min at 10,000 × g, 4°C. Discard supernatant.
10. Wash again using ice-cold isolation buffer 2.
11. Resuspend mitochondria in 0.7 ml isolation buffer 2 and determine protein concen-
tration using a BCA kit (see Support Protocol 2).
12. Adjust to >30 mg protein/ml with isolation buffer 2. Keep on ice and use within 4
to 6 hr.
Mitochondrial Protein Assay SUPPORT

PROTOCOL 2
This protocol describes the quantification of mitochondrial protein using a bicinchoninic
acid (BCA) assay kit.
Assessment of Cell
Toxicity
2.16.9
Materials
2 mg/ml bovine serum albumin (BSA)
1% (v/v) Triton X-100
Mitochondrial sample (see Support Protocol 1)
BCA kit (Pierce) containing protein reagents A and B
2-ml microcentrifuge tubes

Standard 96-well plate
37°C heating block or incubator
Absorbance plate reader
1. Prepare BSA at 1.5, 1.0, 0.75, 0.5, 0.25, 0.125, and 0.025 mg/ml in 1% Triton X-100.
Protein standards can be made in batches and stored for several weeks at 4°C.
2. In 2-ml microcentrifuge tubes, prepare mitochondrial dilutions at 1:60, 1:80, and

1:100 using 1% Triton X-100 as diluent.
3. Pipet 20 µl of each standard and sample into a 96-well plate.
4. Prepare developing reagent by mixing 9.8 ml protein reagent A with 200 µl protein
reagent B.
The solution will turn green.
5. Add 200 µl developing reagent to each well and incubate 30 min at 37°C.
6. Read absorbance at 520 nm.
7. Calculate mitochondrial protein concentration based on comparison of sample values
with the standard curve.
SUPPORT Instrument Signal Check

PROTOCOL 3
This protocol is used to optimize plate reader parameters when using either the
MitoXpress-Xtra or pH-Xtra kit.
Materials
Probe kit (select one):
MitoXpress-Xtra kit, including high-sensitivity (HS) mineral oil
pH-Xtra kit, including respiration buffer tablet
Probe diluent: water, PBS, or culture medium
Culture medium
Time-resolved fluorescence plate reader with temperature control and kinetic

analysis software
96-well plate, black with clear bottom
Multiblock heater (VWR, cat. no. 12621-108)
1. Warm the plate reader to the measurement temperature and prepare the kinetic mea-
surement protocol with the appropriate settings to read at 2- to 3-min intervals over
30 min.
See Table 2.16.1 for recommended instrument settings. Three discrete fluorescence modal-
ities can be used, depending on plate reader: intensity measurement (basic), time-resolved
fluorescence (TR-F) measurement (standard), and dual-read ratiometric TR-F mea-
Fluorescent Assay surement (lifetime calculation) (advanced). See Background Information for additional
for Mitochondrial
Function discussion.
2.16.10
2. Reconstitute MitoXpress-Xtra in 1 ml water, PBS, or culture medium by gently
aspirating three to four times. For pH-Xtra, reconstitute in 1 ml water.
Reconstituted probe can be stored in the dark for several days at 2° to 8°C or up to
1 month in water at −20°C. Repeated freezing and thawing should be avoided.
3. In a 96-well plate on a prewarmed multiblock block heater, add 150 µl prewarmed

culture medium (for MitoXpress-Xtra) to eight replicate wells (A1-A4, B1-B4). For
pH-Xtra, use respiration buffer.
4. Add 10 µl reconstituted probe to wells A1-A4 and add 10 µl probe diluent to wells
B1-B4.
5. For MitoXpress-Xtra, use a syringe dispenser to quickly add 100 µl prewarmed
mineral oil to all eight wells, taking care to avoid air bubbles.
Mineral oil is not necessary with pH-Xtra.
6. Insert microplate into the plate reader and commence reading.

7. When measurement cycle is complete, remove plate from instrument and save data
to file.
8. Examine readings of signal controls (A1-A4) and blank controls (B1-B4) after sig-
nal stabilization and calculate the signal-to-blank (S:B) ratio. For dual-read TR-F
measurements, calculate S:B for each measurement window.
Basic analysis: Prompt fluorescence intensity measurement can be performed on a very
wide range of commonly available fluorescence, monochromator, or filter-based plate
readers. Optimal wavelengths are 380 nm excitation and 650 nm emission, with detection
gain parameters (PMT) typically set at medium or high. MitoXpress-Xtra should return a
S:B 3.
Standard analysis: Increased levels of performance can be achieved by using TR-F, which
reduces nonspecific background and increases probe sensitivity. Optimal delay time is
30 μsec and gate (integration) time is 100 μsec. MitoXpress-Xtra should return a S:B
3, and an S:B of 10 is typical.
Advanced analysis: Dual-read TR-F (lifetime) can be performed on instruments indicated
in Table 2.16.1 and is the preferred option when available. Optimal dual-delay and gate
(integration) times are 30 μsec delay (D1), 30 μsec measurement (W1), 70 μsec delay
(D2), 30 μsec measurement (W2). MitoXpress-Xtra should return a S:B 5. An S:B up to
60 is possible.
For pH-Xtra, alternative TR-F measurements are recommended (see Basic Protocol 2 and
Critical Parameters).
MEASUREMENT OF METABOLIC PERTURBATION IN WHOLE CELLS

Although measuring the impact of drug treatment on isolated mitochondria is a very
useful method for assessing drug-induced mitochondrial toxicity, it can occasionally
over-predict drug-induced toxicity, because test compounds have free access to various
targets within the mitochondria. Microtiter plate–based measurement of cellular oxy-
gen consumption circumvents this issue while the contribution of transporter and CYP
activity can also be incorporated. Cell-based measurements of changes in oxygen con-
sumption immediately post-treatment are therefore a useful tool for further investigation
of drug-induced mitochondrial dysfunction (Hynes et al., 2006; see Basic Protocol 2).
An additional layer of information can be added by assessing glycolytic flux based on
extracellular acidification (see Basic Protocol 3). Measurement of extracellular acidifica-
tion provides data on the rate of conversion of pyruvate to lactic acid, and is therefore a
convenient measure of glycolytic activity (Hynes et al., 2009). Together, these parameters Assessment of Cell
Toxicity
allow an analysis of the interplay between glycolytic and oxidative metabolism, and how
2.16.11
a given drug treatment perturbs this balance (Hynes et al., 2013). Additional information
can be generated by multiplexing with mitochondrial membrane potential (MMP) mea-
surements. In addition, this section includes a protocol for the use of permeabilized cells
(see Support Protocol 4) to facilitate a more mechanistic cell-based analysis.
BASIC Cell-Based Measurement of Oxygen Consumption

PROTOCOL 2
This protocol describes measurement of cellular oxygen in 2D and 3D cultures using
MitoXpress-Xtra, as well as multiplexing with MMP measurements.
Materials
Cells to be tested
Growth medium supplemented for normal growth
MitoXpress-Xtra HS Oxygen Consumption Assay kit (Luxcel Biosciences, cat. no.
MX-200), including high-sensitivity (HS) mineral oil
Test compounds and appropriate solvents
Standard 37°C, 5% CO2 , humidified incubator
Time-resolved fluorescence plate reader (Table 2.16.1) with temperature control
and kinetic analysis software
37°C water bath
50-ml plastic tube
Aspirator or microplate centrifuge
Multichannel or repeat pipetter
Prepare cells
1. Culture cells of interest as follows:
a. For adherent cells: Plate in growth medium in a 96-well plate, typically at 40,000
to 80,000 cells/well. Include blank control wells without cells in each plate.
Incubate at 37°C overnight.
b. For suspension cells: Culture enough cells to achieve the desired number of test
wells at a final density of 4 × 106 cells/ml at the time of the assay. (This typically
requires cells to be concentrated from the culture flask concentration.)
Longer culture periods can be used.
As an initial step, a serial dilution of cells is recommended to determine the optimum
seeding density.
When plating 3D cultures, prepare the 3D plate or 3D construct solution (collagen
reaction mix) in advance according to manufacturer’s instructions. Cells are typically
plated at higher concentrations than for 2D cultures (1.5- to 2-fold higher than the
maximum density for 2D culture). Lower plating concentrations are typically used with
long culture times unless cells are terminally differentiated.
Measurement of cellular oxygen consumption in microtiter plate format requires the
generation of an oxygen consumption rate that overcomes the back diffusion of oxygen
through the body of the plate.
If multiplexing with MMP probes such as JC-1, pre-load cells per manufacturer’s in-
structions. Measure plate as described below.
Fluorescent Assay
for Mitochondrial
Function
2.16.12
appropriate settings to read the desired wells at 0.5- to 1.5-min intervals over 60 to
80 min (Table 2.16.1).
Support Protocol 3). Three discrete fluorescence modalities can be used, depending on
the plate reader: intensity measurement (basic), time-resolved fluorescence (TR-F) mea-
surement (standard), and dual-read ratiometric TR-F measurement (lifetime calculation)
(advanced). See Background Information for additional discussion.
Oxygen consumption is temperature-sensitive, with maximal consumption typically ob-
served at 37°C. Systematic temperature variation within the measurement chamber can
therefore cause a temperature-dependent plate effect. This effect is instrument-dependent
and can be reduced by measuring at 30°C.
If multiplexing kinetically with MMP probes, ensure second measurement label is pre-
pared per manufacturer’s instructions. Alternatively, endpoint measurements can be
performed subsequent to the MitoXpress-Xtra assay.
3. Reconstitute MitoXpress-Xtra probe in 1 ml water and warm to 37°C.
The standard probe package is designed for one 96-well plate. Reconstituted probe can
be stored undiluted in the dark for several days at 4°C. Probe further diluted in growth
medium should be used on the same day.
4. Prepare the test compound dilution series in the appropriate solvent.

To reduce the volumes required, a 96-well PCR plate can be used.
5. Add 16 ml growth medium to a 50-ml plastic tube and warm to 37°C.

6. Warm mineral oil to 37°C.
Set up assay plate
7. Wash cells with fresh, prewarmed growth medium.
a. For adherent cells: Remove medium using an aspirator, being careful not to
dislodge cells. Use a multichannel or repeat pipetter to add 140 µl medium to
each well.
b. For suspension cells: Centrifuge cells 5 min at 150 × g, room temperature.
Remove medium and resuspend cells in fresh medium at a density of 4 × 106
cells/ml. Add 140 μl suspension (5.6 × 105 cells) to each well of a 96-well test
plate.
Include blank control wells containing medium without cells in each plate.
8. Place plate on a plate heater equilibrated to 37°C.
9. Add 10 µl reconstituted MitoXpress-Xtra probe to each well. Include blank control
wells with cells and growth medium but no probe.
10. Add 1 µl test compound to each well (n = 4 is recommended).
If accurate pipetting of 1 µl is not feasible and technical replicates are being used,
compounds can be prepared in bulk growth medium stocks containing MitoXpress-Xtra
probe. These stock are added to test wells after spent medium is aspirated. They can be
store up to 1 week at −20°C. Assessment of Cell
Toxicity
2.16.13
Figure 2.16.3 Sample oxygen consumption data output (lifetime scale) from stem-cell-derived hepatocytes (hiPS-HEP
cells). (A) Z factor analysis showing respiring cells (columns 1-6) and inhibited cells (columns 7-12). (B) Slopes calculated
from A showing the consistency and separation of positive (respiring) and negative (inhibited) samples, yielding a Z factor
of 0.7. (C) Impact of a panel of ETC modulators on hiPS-HEP cell respiration. Measured immediately post treatment,
antimycin, rotenone, and oligomycin cause immediate and complete inhibition, while the uncoupler FCCP causes an
immediate increase on oxygen consumption.
11. Using a syringe dispenser, quickly add 100 µl prewarmed mineral oil to each well,
taking care to avoid air bubbles.
12. Insert the microplate into the prewarmed plate reader and commence reading.
13. When the measurement cycle is complete, remove plate from the instrument and
save data to file.
14. Using the instrument’s data analysis software and templates, plot probe signal pro-
files against time for each well. Select the linear portion of the profile and apply linear
regression analysis to determine the slope for each profile and tabulate appropriate
average and standard deviation values.
Lifetime-based measurement is the preferred mode where available. Lifetime calculations
Fluorescent Assay are performed automatically when using data analysis tools such as those from BMG
for Mitochondrial Labtech and BioTek. These tools will also perform slope calculation and basic statistical
Function
analysis. If such tools are not available, export data to a suitable data analysis package.
2.16.14
Figure 2.16.4 Mechanistic analyses. (A) Oxygen consumption of digitonin-permeabilized succinate-driven HepG2
cells. Unpermeabilized cells show no consumption, as succinate is cell membrane impermeable. Permeabilization
provide succinate access to the mitochondrial network, causing significant oxygen consumption. Rotenone has no
effect as it inhibits the ETC upstream of complex II. Antimycin causes inhibition, as it inhibits downstream of complex
II. (B) Oxygen consumption of JC-1-loaded A549 cells showing uncoupling (FCCP) and inhibition (antimycin) of
oxygen consumption. (C) Multiplexed JC-1 analysis showing an associated collapse in MMP.
Lifetime can be calculated manually as: 40/ln(R1/R2), where R1 and R2 represent read
one and read two. See Background Information for additional discussion.
If ‘drift’ is observed in the relative fluorescence signal (RFU) of negative control wells
(probe but no cells), subtract this value from all test values.
If multiplexing with MMP probes, process data as per manufacturer’s instructions.
See Figures 2.16.3 and 2.16.4 for sample results.
Cell Permeabilization SUPPORT

PROTOCOL 4
This protocol describes digitonin permeabilization of adherent cell monolayers in mi-
crotiter plates, prior to measurement of oxygen consumption.
Additional Materials (also see Basic Protocol 2)
20 mM ADP
3 mg/ml digitonin
75 µM rotenone (150×)
150 μM antimycin A (150×)
100 mM succinate (10×)
Prepare cells, instrument, and reagents
1. Plate cells as described (see Basic Protocol 2, step 1a).
2. Prepare the plate reader as described (see Basic Protocol 2, step 2), ensuring that it
is prewarmed and that settings have been optimized.
3. Prepare one 50-ml plastic tube containing 40 ml measurement buffer 2, and another
50-ml tube containing 17 ml measurement buffer 2 plus 1.0 ml of 20 mM ADP.
4. Reconstitute MitoXpress-Xtra in 1 ml water or measurement buffer 2 by gently
aspirating three to four times.
5. Warm the following reagents to 37°C:
Measurement buffer 2, with and without ADP
Reconstituted MitoXpress-Xtra Assessment of Cell
Toxicity
3 mg/ml digitonin
2.16.15
150 μM antimycin A
75 μM rotenone
100 mM succinate
Mineral oil.
As an example, antimycin A and rotenone are used as test compounds, and 10 mM succi-
nate is used as substrate. Additional ETC substrates such as pyruvate/malate (CI) duro-
quinol (CIII), and TMPD/ascorbate (CIV) can be used to further delineate ETC activity.
Permeabilize cells
6. Aspirate culture medium from plated cells and wash with 100 µl prewarmed mea-
surement buffer 2, being careful not to dislodge the cells. Repeat wash.
7. Add 100 µl measurement buffer 2 to each well.
8. Add 1 μl of 100× digitonin to designated wells, leaving some wells without digitonin
as a control. Gently rock the plate to mix.
To reduce variation due to pipetting, steps 7 and 8 can be combined using a master mix
of 1× digitonin in buffer.
9. Incubate 10 min at 37°C.
Perform assay
10. Aspirate solution and carefully wash cells with measurement buffer 2 + ADP.
Aspirate again and add 140 µl measurement buffer 2 + ADP to each well.
11. Place plate on a plate heater equilibrated to 37°C.
12. Add 10 µl reconstituted MitoXpress-Xtra probe to each well.
13. Using a repeat pipetter, add 1 µl each of 150 μM antimycin A and 75 μM rotenone
to the designated wells.
14. Add 17 μl of 100 mM succinate (final 10 mM substrate).
As succinate is cell membrane impermeable, the combination of succinate and oxygen
consumption also facilitates optimization of the digitonin concentration needed to achieve
permeabilization without causing nonspecific cell damage. This is achieved by assessing
a range of digitonin concentrations and picking the concentration that provides maximal
oxygen consumption. Excessively high concentrations will cause cell damage.
15. Complete as described (see Basic Protocol 2, steps 11-14).
See Figure 2.16.4A for sample results.
BASIC Cell-Based Measurement of Glycolytic Flux
PROTOCOL 3
This protocol describes how glycolytic flux can be interrogated by measuring extracellular
acidification of 2D or 3D cultures using pH-Xtra, and how these measurements can
be multiplexed with MitoXpress-Xtra-based oxygen consumption measurements. The
procedure is similar to Basic Protocol 2, details of which are cited below.
Materials
Cells to be tested
Growth medium supplemented for normal growth
pH-Xtra Glycolysis Assay kit (Luxcel Biosciences, cat. no. PH-200), including
Fluorescent Assay respiration buffer tablet
for Mitochondrial Test compounds and appropriate solvents
Function
2.16.16
Standard 37°C, 5% CO2 , humidified incubator
CO2 -free, 37°C, 95% humidified incubator
pH meter
Time-resolved fluorescence plate reader (Table 2.16.1) with temperature control
and kinetic analysis software
0.22-µm syringe filter
37°C water bath
Aspirator or microplate centrifuge
Prepare cells
1. Grow adherent or suspension cells as described (see Basic Protocol 2, step 1).
2. Transfer cells to a CO2 -free incubator 2.5 hr prior to measurement.
When culturing cells in a standard 5% CO2 environment, the polystyrene body of a
standard microtiter plate absorbs CO2 . If the plate is moved directly to the plate reader,
this CO2 will diffuse into the sample, causing significant acidification. Thus, cells are
placed in a CO2 -free environment prior to measurement to allow the CO2 to diffuse
away. Alternatively, cells can be cultured in CO2 -free conditions using a specially
formulated medium such as L-15.
3. Warm the plate reader to 37°C and prepare the kinetic measurement protocol as
described (see Basic Protocol 2, step 2).
In this case, two fluorescence modalities can be used: standard (TR-F) or advanced (dual-
read ratiometric TR-F, lifetime calculation). Lifetime measurements are recommended
where available, allowing subsequent conversion to pH and [H+ ] scales. See Background
Information for additional discussion.
Like oxygen consumption, glycolytic flux and pH are temperature-sensitive, and
temperature-dependent plate effects can be reduced by measuring at 30°C.
If measuring oxygen depletion in parallel, prepare a second measurement label as de-
scribed (see Basic Protocol 2, step 2).
4. Reconstitute a respiration buffer tablet in 50 ml water and adjust to pH 7.4. Filter
sterilize using a 0.22-μm filter and warm to 37°C.
Unbuffered DMEM (measurement buffer 3; see recipe) can also be used.
5. Reconstitute the transparent contents of a pH-Xtra vial in 1 ml respiration buffer,
gently aspirating three to four times. Warm to 37°C.
The standard probe package is designed for one 96-well plate. Probe reconstituted in
buffer should be used on the same day.
If measuring oxygen depletion, prepare MitoXpress-Xtra probe (see Basic Protocol 2,
step 3), but use pH-Xtra respiration buffer.
6. Prepare test compounds as described (see Basic Protocol 2, step 4).
If measuring oxygen depletion, also prewarm mineral oil.
Set up assay plate
7. Wash cells with prewarmed respiration buffer.

Assessment of Cell
Toxicity
2.16.17
Figure 2.16.5 ECA measurements of 2D and 3D cultures showing the impact of glucose addition (A,B) and drug
treatment (C,D) on metabolic activity. (A) pH-Xtra data output (lifetime scale) from HepG2 cells in 5.5, 0.5, and 0 mM
glucose. (B) Raw ECA profiles such as those in A transposed to H+ scale, showing the impacts of increasing glucose
concentration and oligomycin treatment on glycolytic flux. (C) ECA profiles from HepG2 cells seeded in collagen-based
3D RAFT cultures. Antimycin and rotenone treatments increase ECA and oxamate treatment causes a reduction. (D)
Raw ECA profiles transposed to H+ scale, expressed as a percentage of untreated values (purple) and compared
to a parallel oxygen consumption rate measurement (grey). The uncoupler FCCP causes an increase in oxygen
consumption and a compensatory increase in glycolytic flux as indicated by the increased ECA. The inhibitors rotenone
and antimycin inhibit mitochondrial function and also cause a compensatory increase in glycolytic flux.
a. For adherent cells: Remove medium using an aspirator, being careful not to
dislodge cells. Use a multichannel or repeat pipetter to add 150 µl buffer to each
well. Repeat, but add 140 μl buffer/well for the second wash.
b. For suspension cells: Centrifuge cells 5 min at 150 × g. Remove medium and
resuspend cells in buffer at a density of 2 × 106 cells/ml. Plate 140 μl suspension
(3 × 105 cells) per well.
Include blank control wells containing buffer without cells in each plate.
8. Place the plate on a plate heater and add 10 μl reconstituted pH-Xtra probe followed
by 1 μl test compound to each well (see Basic Protocol 2, steps 8-10). Include blank
control wells with cells and buffer but no probe.
If measuring oxygen depletion, set up the plate with replicate halves, adding pH-Xtra to
one half and MitoXpress-Xtra to the other. Add prewarmed mineral oil to wells containing
MitoXpress-Xtra (see Basic Protocol 2, step 11).
Fluorescent Assay As with MitoXpress-Xtra, if accurate pipetting of 1 µl is not feasible and technical
for Mitochondrial
Function replicates are being used, compounds can be prepared in bulk stocks containing pH-Xtra
probe and added to test wells after spent medium is aspirated.
2.16.18
9. Acquire and save data (see Basic Protocol 2, steps 12-13).
10. Using the instrument’s data analysis software and templates, plot probe intensity or
lifetime profiles against time for each well. Select the linear portion of the profile and
apply linear regression analysis to determine the slope for each profile and tabulate
appropriate average and standard deviation values.
See Basic Protocol 2, step 14, for additional details. The tools described will also convert
into pH scale. Alternatively, after manual lifetime calculation, the data can be converted
to pH units as follows: pH = (1759.5 − τ )/214.07. These data can then be converted to
[H+] values, facilitating more accurate dose-response data.
If measuring oxygen depletion, process oxygen data as described.
See Figure 2.16.5 for typical results.
Antimycin A, rotenone, and FCCP
1 mg/ml glucose oxidase (GOx)
MEASURING CELLULAR OXYGENATION AND ASSOCIATED

METABOLIC SHIFTS
Knowledge of in situ oxygenation of cells in 2D and 3D cultures offers important insights
into the impact of oxygen on cell metabolism and, in turn, how a cell responds to toxic in-
sult. In addition, focus is also now shifting to the culture and testing conditions used, with
a growing desire to perform such analysis under more physiologically relevant conditions
by providing relevant respiratory substrates and biologically relevant oxygen concentra-
tion, as opposed to the supraphysiological glucose and oxygen concentrations often used.
Measurement of Cellular Oxygenation Using Fluorescent Intracellular Oxygen BASIC

Probes PROTOCOL 4
The following protocol outlines how such measurements can be performed in 2D and 3D
cultures using conventional time-resolved fluorescence plate readers and the intracellular
oxygen-sensitive probe MitoXpress-Intra. The protocol also illustrates how the impact of
drug treatment on cell oxygenation can be assessed and how the link between oxygenation
and glycolytic metabolism can be examined.
Materials
Cells to be tested
Culture medium
MitoXpress-Intra (Luxcel Biosciences, cat. no. MX-300)
Test compounds and appropriate solvents
96-well plate, black with clear bottom
Standard CO2 incubator
CO2 -free, 37°C, 95% humidified incubator
37°C water bath
Aspirator
Appropriate time-resolved fluorescence plate reader (Table 2.16.1)
50-ml plastic tube
Assessment of Cell
Toxicity
2.16.19
Plate cells
1. Count cells and adjust to a plating density that gives the desired number of cells in
200 μl culture medium (typically 3 × 105 cells/ml to give 6 × 104 cells/well
for 2D cultures). Plate 200-μl aliquots of cells in all but the outer wells of a 96-well
plate. Include a probe control without cells in each plate.
Cells are typically plated at a density to achieve full confluence. Plating density and
basal metabolic rate will determine the steady-state oxygen concentration at the cell
monolayer.
When plating 3D cultures, prepare the 3D plate or 3D construct solution (collagen
reaction mix) in advance as per the manufacturer’s instructions. Cells are typically
plated at higher concentrations than used for 2D cultures. Lower plating concentrations
are typically used with long culture times unless cells are terminally differentiated.
2. Add 200 µl culture medium to the outer wells of the plate to reduce possible
evaporation and improve temperature consistency across the plate.
3. Place plate in a CO2 incubator overnight (typically >14 hr).
Load cells
4. Reconstitute MitoXpress-Intra in 11 ml culture medium and warm to 37°C.
The standard probe package is designed for one 96-well plate. Probe diluted in medium
should be used within 24 hr.
5. Place the plate containing cells on a multiblock heater equilibrated at 37°C. Remove
medium using an aspirator, being careful not to dislodge the cells.
The multiblock heater helps maintain the temperature of the plate and minimizes warm-
up time once measurement is started. To prevent cells from drying out, do not aspirate
more than 20 wells at a time.
6. Using a multichannel or repeat pipetter, add 100 µl prewarmed MitoXpress-Intra to
each well, being careful not to dislodge the cells. Return the plate to the incubator
overnight (typically > 14 hr).
Loading can also be performed directly in T75 cm2 flasks using the same probe con-
centration and incubation time.
Shorter incubation times with a higher probe concentration can be used if necessary.
For example, a 6-hr loading time at 1.5× probe concentration will yield comparable
loading and baseline fluorescence. This may be dependent on cell type, so optimization
is recommended if probe loading conditions are changed.

appropriate settings to read the desired wells at 2- to 3-min intervals over the
desired duration (Table 2.16.1).
Support Protocol 5). Dual-read ratiometric TR-F measurement (lifetime calculation) is
recommended where available. See Background Information for additional discussion.
8. If measuring at defined O2 and CO2 concentrations, set the atmospheric control

unit according to manufacturer’s instructions.
A plate reader with an atmospheric control unit allows 5% CO2 to be applied to the
measurement chamber.
Fluorescent Assay
for Mitochondrial
Function
2.16.20
9. Add 40 ml measurement buffer 4 to a 50-ml plastic tube and warm to 37°C.
Measurement using standard cell culture medium is possible for short-term experiments
(<2 hr) in CO2 -free conditions. Measurement buffer 4 allows longer-term measurement
(>2 hr) outside a CO2 incubator (in a plate reader), as it is capable of maintaining pH
for a prolonged period without applied CO2 .
10. Prepare test compound(s) such as antimycin, rotenone, and FCCP in the appropriate
solvent according to the required plate map.
Typically, 150× stock concentrations are recommended in order to minimize the final
concentration of DMSO (or other solvents) in the assay mix (0.6% v/v).
Set up assay plate

11. Return plate to the multiblock heater and remove probe solution using an aspirator,
being careful not to dislodge the cells.
12. Using a multichannel or repeat pipetter, add 100 µl prewarmed measurement buffer
4 to each well. Repeat this wash step again.
13. Aspirate again and add 150 µl prewarmed measurement buffer 4 to each test well.
Add 150 µl measurement buffer 1 to the blank control wells.
Perform measurement
For endpoint oxygenation assay with compound treatment(s)
14a. Add 1 µl of 150× test compound to the desired test wells and return the plate to
the incubator for the required incubation period. Add 10 μl of 1 mg/ml GOx to
designated wells to provide complete sample deoxygenation.
15a. Insert plate into the plate reader and commence endpoint reading.
The endpoint protocol should include an incubation period to allow temperature equi-
libration, drug action and re-equilibration of intracellular oxygen concentrations. The
time required is dependent on the culture system being used and the biological processes
under investigation, but 60 min is typical.
16a. When the measurement cycle is complete, remove the plate and save data to file.
For kinetic assay with compound treatment(s)

14b. Insert plate into the plate reader and commence kinetic reading. Allow the baseline
signal to be measured for at least 20 min.
15b. Pause the measurement and deliver 1 μl of 150× test compound to the desired
wells. Add 10 μl of 1 mg/ml GOx to designated wells to provide complete sample
deoxygenation.
Additions can be made using the plate reader’s injectors or (if injectors are unavailable)
by ejecting the plate from the reader, quickly adding test compound to the desired
wells, and re-inserting the plate into the plate reader. The time that the plate is out
of the measurement chamber should be kept to a minimum (>2 min), particularly if
measurements are being carried out in low O2 .
16b. Resume kinetic measurement.
After responses are observed and the signal has re-stabilized, further compound addi-
tions can be made.
As the assay is nondestructive, the plate can be used for subsequent analyses, such as Assessment of Cell
protein content, cellular ATP content, or lactate dehydrogenase (LDH) release. Toxicity
2.16.21
Figure 2.16.6 Sample data output for monitoring cellular oxygenation using MitoXpress-Intra.
(A) Analysis of applied O2 concentration and resultant intracellular O2 concentration in a 3D
HepG2 culture (RAFT, Lonza). Data transposed to oxygen scale. (B) Difference between applied
and intracellular O2 concentrations across a range of applied O2 concentrations, illustrating the
impact of respiration on local oxygen concentrations. (C) The impact of applied O2 concentra-
tion on glycolytic flux, with reduced oxygen availability causing concomitant increases in ECA.
Data generated on a FLUOstar Omega (BMG Labtech) with integrated atmospheric control and
automatic oxygen scale data output.
17b. When the measurement cycle is complete, remove the plate and save data to file.
Analyze data
18. Use the instrument’s data analysis software and templates to plot oxygenation
profiles against time for each well and determine appropriate average and standard
deviation values.
For end-point measurements, single point oxygenation values are generated.
Lifetime calculations and conversions to oxygen scale are performed automatically
when using data analysis tools such as those from BMG Labtech and BioTek. If such
tools are not available, export to a suitable data analysis package. Lifetime can be
calculated manually using blank corrected intensity values as follows 40/ln(R1/R2),
where R1 and R2 represent read one and read two. This provides lifetime values with
units of μsec. Lifetime profiles can be converted in an oxygen scale using the supplied
default calibration function (O2 % = 522.8 × (− τ / 9.064). For maximum accuracy,
dedicated calibrations can be developed (Support Protocol 5).
Measurements can be multiplexed with pH-Xtra facilitating parallel analysis of cellular
oxygenation and glycolytic flux.
See Figure 2.16.6 for typical results.
Fluorescent Assay
for Mitochondrial
Function
2.16.22
Oxygen Calibration of the Fluorescence Plate Reader Using MitoXpress-Intra SUPPORT
PROTOCOL 5
Oxygen calibration can be performed either using a suitable fluorescence plate reader
housed in a hypoxic environment or using a fluorescence plate reader with a pre-installed
atmospheric control unit. Both approaches use nitrogen gas to modulate the level of oxy-
gen within the measurement chamber. Calibration is performed under defined conditions
(temperature, assay volume, instrument parameters) across multiple applied oxygen con-
centrations, typically starting at atmospheric conditions (room air) and gradually reducing
the applied oxygen concentration in a stepwise manner over time. Data are generated
from samples treated with 1 µM of the ETC inhibitor antimycin A. This removes the
influence of cell respiration on intracellular oxygen concentrations, so the measured sig-
nal reflects only the applied oxygen concentration, allowing construction of a calibration
function. Additional wells are treated with glucose oxidase, which completely removes
oxygen from the sample so the measured signal reflects complete deoxygenation.
Additional Materials (also see Basic Protocol 4)
150 μM antimycin A
1 mg/ml glucose oxidase (GOx)
1. Culture, plate, and load cells as described (see Basic Protocol 4, steps 1-6).
2. Prepare the plate reader as described (see Basic Protocol 4, steps 7-8), ensuring that
it is prewarmed, that settings have been optimized, and that the oxygen concentration
has equilibrated to room air conditions (21% O2 ).
3. Prepare reagents and set up the assay plate as described (see Basic Protocol 4, steps
9-13), with the following additions:
a. Add 1 µl of 150 μM antimycin A to designated wells to inhibit cellular oxygen

consumption.
b. Add 10 µl of 1 mg/ml GOx to designated wells to provide complete sample
deoxygenation.
4. Perform kinetic measurement as described (see Basic Protocol 4, steps 14b-17b),
reducing the applied oxygen concentration in a stepwise manner. After each reduction,
measure probe signal for 30-45 min or until the signal reaches a plateau.
A typical oxygen concentration series is 20%, 15%, 10%, 7.5%, 5%, 2.5%, and 1% O2 .
An eight- to ten-point calibration is recommended.
5. Use the instrument’s data analysis software to plot oxygen concentration against
measured emission lifetime.
Lifetime calculations and conversions are performed automatically when using data analy-
sis tools such as those from BMG Labtech and BioTek. If such templates are not available,
export to a suitable data analysis package. Lifetime can be calculated manually using
blank-corrected intensity values as follows: 40/ln(R1/R2), where R1 and R2 represent
read one and read two. This provides lifetime values with units of μsec.
REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent in all recipes and protocol steps, and store all
solutions in glassware that has been rinsed with Milli-Q-purified water. For common
stock solutions, see APPENDIX 2A.
Isolation buffer 1
210 mM mannitol
70 mM sucrose
Assessment of Cell
Toxicity
continued
2.16.23
5 mM HEPES
1 mM EGTA
Adjust pH to 7.4 using HCl or KOH
Store up to 1 week at 4°C
On the day of the experiment, add 0.5% (w/v) BSA
Do not use NaOH to adjust the pH.
Isolation buffer 2
210 mM mannitol
70 mM sucrose
10 mM MgCl2
5 mM K2 HPO4
10 mM MOPS
1 mM EGTA
Measurement buffer 1
250 mM sucrose
15 mM KCl
1 mM EGTA
5 mM MgCl2
30 mM K2 HPO4
30 mM HEPES
5 mM potassium phosphate
40 mM sucrose
0.5 mM EDTA
3 mM MgCl2
Sterilize using a 0.2-μm filter
Store up to 1 month at 4°C
Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, cat. no. D5030)
1.85 g/liter NaCl
10 ml 100× GlutaMax
10 ml 100 mM sodium pyruvate
15 mg phenol red
25 mM glucose
Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, cat. no. D5030)
20 mM HEPES
1 mM sodium pyruvate
Fluorescent Assay 20 mM glucose
for Mitochondrial
Function continued
2.16.24
10% FBS
100 U/ml penicillin
100 mg/ml streptomycin
Sterilize using a 0.2-μm filter
Store up to 1 month 4°C
COMMENTARY
Background Information gen consumption. In control samples (no
cells/mitochondria), or in samples where ETC
Oxygen consumption and the ETC
activity has been completely inhibited, the
Measurement of cellular oxygen consump-
probe signal remains stable, as no oxygen is
tion is essentially a measure of the activity of
being consumed.
cytochrome c oxidase, complex IV of the elec-
tron transport chain (ETC). Reduced oxygen
consumption indicates reduced electron flux Extracellular acidification and glycolysis
through the ETC, which can be due to a num- Glycolytic flux may be assessed through
ber of factors including control mechanisms analysis of the rate at which cells acidify their
such as elevated MMP, inhibition of an individ- extracellular environment. This extracellular
ual complex of the chain, or a reduction in the acidification is due to both the conversion of
supply of reducing equivalents. Increased oxy- pyruvate to lactic acid, allowing the anaero-
gen consumption is indicative of uncoupling, bic regeneration of NAD+ , and the production
whereby ETC activity is uncoupled from ADP of CO2 through the Krebs cycle. At physio-
phosphorylation by dissipation of the MMP. logical pH, lactic acid is dissociated into H+
MitoXpress-Xtra is based on an oxygen- and lactate. As intracellular pH is tightly reg-
sensitive metalloporphyrin and has excitation ulated, the H+ produced is extruded into the
maxima at 380 nm and 535 nm and an emis- extracellular environment through a variety of
sion maximum of 650 nm. It exhibits emission transport mechanisms. In an unsealed system,
lifetimes in the microsecond range (26 to the pyruvate-to-lactate conversion is the main
67 μsec on deoxygenation), thereby facil- contributor to extracellular acidification, par-
itating the use of conventional time-resolved ticularly for transformed cell lines, allowing it
fluorescence measurements to increase as- to be used as a measure of glycolytic flux.
say performance. Probe emission (phospho- pH-Xtra is based on a hydrophilic long-
rescence) is quenched by molecular oxygen decay pH-sensitive lanthanide with an excita-
via a physical (collisional) mechanism such tion maximum at 370 nm and three line-type
that, under normal conditions, the oxygen dis- emission bands at 590, 615, and 695 nm. It
solved in the sample partially quenches probe exhibits emission lifetimes in the microsec-
emission. As oxygen is consumed by cellu- ond range. Emission is modulated by the pH
lar/mitochondrial activity, the concentration of of the sample (214 to 450 μsec, moving
dissolved oxygen in the well is reduced, re- from pH 7.4 to 6.4), allowing acidification to
sulting in an increase in probe signal. An addi- be detected as an increase in probe emission.
tional consideration is that, as dissolved oxy- Lifetime measurement (ratiometric TR-F) pro-
gen is consumed, the oxygen gradients formed vides the best performance and is achieved
drive the back diffusion of ambient oxygen on time-resolved plate readers by measuring
at the liquid-air interface and through the probe intensity at two delay times following
polystyrene body of the microtiter plate. Suc- successive intensity pulses, and using the ratio
cessful analysis requires rates of consumption of these two intensity measurements to calcu-
that exceed the rate of back diffusion. A seal- late an emission lifetime (Hynes et al., 2009).
ing layer of mineral oil limits back diffusion, This provides a more robust indicator of pH
thereby increasing assay sensitivity. Mea- than can be achieved using standard inten-
surement conditions are optimized such that sity measurements. Calculated lifetimes can be
untreated cells/mitochondria give a reliable converted to pH values using a simple trans-
signal change over the measurement period. formation, and these in turn can be converted
Uncoupling causes an increase in the rate to H+ values. Such transformations can be
of signal change due to increased oxygen managed using customized data analysis tools
consumption, while inhibition reduces the available from plate reader manufacturers such Assessment of Cell
rate of signal change due to reduced oxy- as BMG Labtech and BioTek. Measurement Toxicity
2.16.25
conditions are optimized such that untreated provide a robust calibration function allow-
cells give a reliably measurable signal change ing measured lifetime values to be transposed
over the measurement period. Wells contain- into an oxygen scale without the need for
ing cells show an increase in probe signal over on-board calibration. Nonetheless, for max-
time due to continuing extracellular acidifica- imum accuracy, cell and instrument calibra-
tion, while control wells (either wells without tions are recommended. Measurement pro-
cells or wells where glycolysis has been com- vides real-time information on the concentra-
pletely inhibited) show no change in signal tion of molecular oxygen within the cell mono-
(Fig. 2.16.4). layer or 3D structure across multiple samples,
facilitating the analysis of transient changes
Cellular oxygenation and physiological in metabolic activity and providing critical in-
normoxia formation on the levels of oxygen available to
A need for improving the biological rele- the cell model under study, something which
vance of the in vitro cell models used to assess is beyond the capacity of extracellular sensing
drug toxicity has drawn focus on the cell types methodologies.
and culture conditions used for such analysis.
This has led to increased adoption of stem- Critical Parameters and
cell-derived cell types and 3D culture systems. Troubleshooting
In related fields, a trend is also emerging to- Mitochondrial isolation
wards the use of lower, more biologically rel- Low activity or poor respiratory control ra-
evant oxygen concentrations (Chapple et al., tio (RCR) values. This can be caused by using
2016), driven by an appreciation that the air- animals that are too old, contaminated glass-
saturated conditions typically used are hyper- ware (leading to uncoupling), or overly harsh
oxic for most cell types (Toussaint et al., 2011), digestion or homogenization conditions.
the relevance of which is underscored by the Activity diminishes in less than 4 to 6 hr.
impact of oxygenation levels on processes as This can be caused by mitochondria being
diverse as stem cell differentiation and drug stored at <30 mg/ml, not being kept on ice,
resistance development within tumors. How- or being excessively handled.
ever, although control of ambient oxygen con-
centrations is easily achievable, the key in- Assays
formation required is the level of pericellular Signals are indistinguishable from blanks.
oxygenation. To date the lack of in vitro tools Check instrument suitability and setup, and
capable of measuring this parameter has pre- run proper controls with and without probe
vented a detailed understanding of the rela- to ensure that specific probe signal is being
tionship between available oxygen and con- detected.
comitant metabolic alterations that can impact Signals are detectable, but signal changes
cell responses to toxic insult. This is particu- are small. Check the instrument settings and
larly true of stem cell models cultured in 3D, measurement temperature. For oxygen mea-
where oxygen concentrations can be signifi- surements, ensure that the oil seal has been
cantly lower than those applied to the culture applied. If signal changes are still too small,
system. increase cell number or mitochondrial con-
As with MitoXpress-Xtra, the phosphores- centration. To increase cell numbers beyond
cent emission of MitoXpress-Intra is quenched full confluence, culture cells in standard flasks,
by molecular oxygen such that a decrease in then trypsinize and concentrate them prior to
the oxygen concentration causes an increase measurement.
in measured signal. The probe is taken up Initial signal is inconsistent. Ensure that
by endocytosis, whereupon probe signal can probe is added accurately. Using time-resolved
be measured to indicate intracellular oxygen fluorescence mode and employing the ratio-
concentrations. Lifetime-based sensing is fa- metric approach outlined above will also re-
vored for such measurements, as it allows di- duce inconsistency.
rect correlation with absolute oxygen concen- Drug interferes with signal. For mitochon-
trations and provides greater robustness due drial measurements, the recommended data
to the fact that it is independent of variabil- transformation procedure deals with the vast
ity in probe loading, fluctuations in intensity, majority of these effects. For cell measure-
and scattering, all of which can significantly ments, ratiometric (lifetime) measurements
Fluorescent Assay affect standard prompt fluorescence measure- deal with the majority of such effects.
for Mitochondrial ments. Lifetime measurements are achieved in Declining signal over the initial 10 min
Function
the same manner as described for pH-Xtra and of measurement. This is caused by the
2.16.26
plate heating from room temperature to the Instrument signal check
measurement temperature, and can be elimi- It is possible to check signal fold in-
nated by preparing the plate on a plate heater crease. Include additional signal wells contain-
and prewarming all solutions. Alternatively, ing reagent in wells C1-C4, and add 10 µl of
one can conduct data analysis after the control 1 mg/ml GOx solution to these wells.
signal has stabilized.
Storage and stability. MitoXpress-Xtra and Cell permeabilization
pH-Xtra should be stored at 2° to 8°C (see If there is digitonin permeabilization is
“Use Before” date on vial). Reconstituted insufficient, perform a digitonin titration
probe can be stored for several days in the experiment using three-fold serial dilutions
dark at 2° to 8°C, or up to 1 month as aliquots starting with a top concentration 10× the rec-
in water at −20°C (avoid freeze-thaw cycles). ommended digitonin concentration.
Plate reader. The fluorescence plate reader Anticipated Results
must be capable of measuring excitation at
380 nm and emission at 650 nm (O2 ) and ETC activity in isolated mitochondria
615 nm (pH), and must have temperature Due to possible batch-to-batch variability
control. of mitochondrial preparations and/or varia-
Plates. Either 96- or 384-well TC+ plates tions in the source tissue, quality control of
with black walls and clear bottoms are rec- the mitochondrial preparation, optimization of
ommended, although standard clear-wall poly- the working concentration, and evaluation of
styrene plates for cell culture may also be used. assay performance are required prior to the
Temperature. A plate block heater should assessment of effector action. Oxygen con-
be used during plate preparation to maintain a sumption rates are low under basal conditions
temperature of 37°C. The plate reader should (State 2; substrate only) and increase dramati-
be prewarmed to the measurement tempera- cally upon addition of ADP (State 3). The ratio
ture, and all culture media and stock solutions of ADP-stimulated to basal respiration (State
should be warmed prior to use. 3/State 2 or RCR) is a measurement of func-
Signal optimization and use of controls. A tionality (coupling) of the mitochondria and is
signal optimization check is recommended, es- an indication of preparation quality. Full cou-
pecially for first time users. Inclusion of blank pling of mitochondrial preparations should be
and optional additional control wells is also assured prior to compound screening.
important. RCR values are calculated by assessing
Pipetting HS mineral oil. Warm mineral oil the effect of ADP addition. Sample raw pro-
before use and take care when dispensing to files are presented in Figure 2.16.2C, where
avoid bubbles. Apply to the inside surface of ADP addition causes a significant increase in
the well, allowing the oil to run down into the oxygen consumption. Calculated RCR values
well. Do not shake or rapidly aspirate mineral should be compared with those in the literature
oil. (Hynes et al., 2006), previous experiments,
Cell type and cell density. Use as high a cell or control data sets. RCRs will differ for dif-
density per well as practical as a starting point, ferent substrates and are tissue-dependent. As
then reduce cell numbers as required. a guideline, rat liver mitochondria in general
Signal-to-blank (S:B) optimization. Set up produce RCRs of 3.0 for succinate-driven
according to Table 2.16.1 and optimize ac- respiration and 5.0 for glutamate/malate-
cording to Support Protocol 3. Measurements driven respiration. Different substrates can be
should return a S:B ratio 3. The following used in screening paradigms for mechanistic
options may be helpful to improve the S:B ra- purposes.
tio: For drug testing, the optimal protein
1. Increase gain (PMT) setting or flash en- concentrations for each analyzed state and
ergy substrate need to be determined. The op-
2. Adjust TR-F focal height timal working concentration provides rela-
3. Increase length of integration time (the tively large signal changes without induc-
same for both delay windows). ing rapid signal saturation, thus allowing
4. Repeat as top or bottom read, respec- reliable analysis of both uncoupling and in-
tively. hibition of mitochondrial respiration. The op-
5. Increase volume of MitoXpress-Xtra or timal protein concentrations for mitochon-
pH-Xtra (15 μl). dria from Sprague-Dawley rat liver are:
6. Contact instrument supplier for further 0.5 mg/ml for succinate-driven basal respira- Assessment of Cell
Toxicity
options. tion; 0.125 to 0.25 mg/ml for succinate-driven
2.16.27
ADP-activated respiration; 1.0 mg/ml for are similar to those obtained from mitochon-
glutamate/malate-driven basal respiration; and drial assessments, although the signal changes
0.25 mg/ml for glutamate/malate-driven ADP- are usually significantly smaller due to lower
activated respiration. levels of oxygen consumption. Analysis typ-
Assay performance is determined from ically involves a comparison between treated
CV values for intra- and inter-assay varia- and untreated samples and overcomes the lim-
tions. These values should normally should itation of many conventional mitochondrial
be 10% (and should not exceed 15%) be- toxicity assays in that impaired mitochondrial
fore moving on to compound screening. Once function is detectable specifically and imme-
mitochondrial preparations are shown to be diately, uncoupling may be distinguished from
reproducible and the assay is well-established inhibition, ATPase inhibition is detectable, and
(n = 3 to 5), optimization becomes unneces- all these effects may be visualized in real time
sary as long as conditions remain consistent. in conventional microplate format.
When changing parameters such as the ani- Characteristic oxygen depletion profiles
mal species or strain, the tissue source, or the from stem-cell-derived hepatocytes are pre-
treatment, re-optimization is required. sented in Figure 2.16.3. Respiration causes
Mitochondrial toxicity, as measured by oxygen depletion, resulting in an immediate
oxygen consumption, can be a result of un- increase in probe signal (columns 1-6). When
coupling (seen as an increase in oxygen con- respiration is inhibited, no oxygen depletion
sumption) or inhibition (seen as a decrease occurs and thus no signal changes are ob-
in oxygen consumption). Uncoupling is best served (columns 7-12). Slopes obtained from
evaluated during basal respiration (State 2), as such profiles indicate the rate of probe sig-
effects on ADP-stimulated respiration (State nal increase and show screening performance
3) can easily be misinterpreted. Inhibition is (Z = 0.7; Fig. 2.16.3B). These slopes are also
most easily detected during ADP-stimulated used to compare treated and untreated cells
analysis (State 3). For qualitative assessments (see sample data in Fig. 2.16.3C). Uncoupling,
of uncoupling or inhibition, straightforward caused by FCCP addition, results in an in-
analysis of raw fluorescence profiles is often creased rate of oxygen consumption and an
sufficient (Fig. 2.16.2D,E). This allows pre- increased rate of probe signal change. Inhibi-
liminary detection of inhibition or uncoupling tion, caused by antimycin or rotenone, block
and generation of rudimentary dose-response the activity of the ETC, shutting down oxygen
information. Dose-response data and/or com- consumption.
pound ranking is achieved by simple regres- Additional mechanistic information can
sion analysis of such profiles. For more quan- be generated by permeabilizing the cell
titative data, these fluorescence profiles can membrane, facilitating direct delivery of
be converted to oxygen scale as outlined in reducing equivalents to each complex of the
Basic Protocol 2. A recommended compound ETC. This facilitates the interrogation of in-
screening plate map is presented in Figure dividual ETC components without disrupting
2.16.1B, with sample data output presented the mitochondrial network. As an example,
in Figure 2.16.2D and summarized in Figure measurements from digitonin-permeabilized
2.16.2E. Measurements are done in duplicate succinate-driven HepG2 cells are shown in
and inhibitors can be clearly identified. Fig- Figure 2.16.4A. As succinate cannot pene-
ure 2.16.1C shows a typical plate layout for trate the cell membrane, unpermeabilized cells
generating dose-response curves (IC50 /UC50 ). consume no oxygen and therefore show no
Results can then be compared to reference lit- signal change. Permeabilization allows suc-
erature or in-house data. The relatively high cinate to access the mitochondrial network,
throughput of this measurement approach also delivering reducing equivalents to complex II
allows for application in structure-activity re- and causing significant oxygen consumption.
lationship investigations. Rotenone has no effect on this consumption,
indicating that it inhibits upstream of the site
Cell-based oxygen consumption assay of reducing equivalent delivery, whereas an-
Mitochondrial function can also be as- timycin causes complete inhibition, indicat-
sessed in 2D and 3D cell cultures. Mitochon- ing that it inhibits downstream. Additional
dria in these systems are influenced by addi- ETC substrates such as duroquinol (CIII) and
tional factors such as compound permeability, TMPD/ascorbate (CIV) can be used to fur-
Fluorescent Assay biotransformation, and secondary mitochon- ther delineate ETC activity. The combina-
for Mitochondrial dria effects—factors that can be influential in tion of succinate and oxygen consumption
Function
vivo. The data output of such measurements also facilitates optimization of the digitonin
2.16.28
concentration to achieve permeabilization uncoupler FCCP impairs the cells’ abil-
without causing nonspecific cell damage. ity to produce ATP aerobically, causing
Further mechanistic information can be an increase in oxygen consumption and a
generated by multiplexing with other rele- compensatory increase in glycolytic flux to
vant metabolic endpoints; MMP multiplexing maintain cellular ATP. Rotenone and an-
is presented as an example in Figure 2.16.4B. timycin inhibit mitochondrial ATP production,
MitoXpress-Xtra and JC-1 are measured in the as evidenced by a decrease in oxygen con-
same test well, allowing simultaneous analy- sumption, and also cause a compensatory in-
sis of ETC activity and MMP. FCCP causes crease in glycolytic flux. Such parallel mea-
a characteristic collapse in MMP and a re- surements offer a deeper insight into metabolic
sultant increase in O2 consumption, whereas perturbation and can be combined with other
antimycin blocks the ETC and inhibits oxygen metabolically relevant end-points, such as
consumption, and as a result MMP is run down ATP and MMP. Other 3D systems including
by MMP-consuming cellular activities. This Alvetex and Mimetix can also be used.
approach can also provide deeper data density
and more mechanistic information through ki- Cellular oxygenation using intracellular
netic measurement. oxygen probes
MitoXpress-Intra facilitates high-
Extracellular acidification assay for throughput measurement of cellular oxy-
assessment of glycolytic flux genation on TR-F-enabled plate readers,
pH-Xtra assay data output is similar to that circumventing many of the difficulties as-
of a MitoXpress-Xtra assay in that samples sociated with invasive microelectrode-based
containing cells give positive signal enhance- assessments and broadening the range of
ment, the rate of which is reflective of the level measurable biological phenomena (O’Riordan
of metabolic activity. In an un-sealed system, et al., 2007; Sung et al., 2010; Chapple et al.,
acidification is due mainly to lactate produc- 2016). In addition, tissue deoxygenation can
tion and is therefore an indicator of glycolytic be modeled using a plate reader equipped with
flux (Hynes et al., 2009). Data output is typ- an atmospheric control unit. In Figure 2.16.6,
ically in lifetime scale, and the data can be sample data are presented illustrating that the
transposed into H+ scale, with rates of acidi- oxygen concentration within metabolically
fication calculated by simple linear regression. competent monolayers or 3D structures can
Sample data describing the effect of glucose deviate significantly from the applied oxygen
concentration on acidification of 2D HepG2 concentration, particularly at physiologically
cultures are presented in Figure 2.16.5A. No relevant oxygen concentrations. This can
signal change is observed in the absence of glu- significantly impact the balance between
cose, but significant acidification is observed oxidative and glycolytic ATP-generating
is the presence glucose. These raw profiles pathways, which in turn can impact the
can be assessed in pH or H+ scale; H+ -based response of a cell model to toxic insult.
acidification is presented in Figure 2.16.5B. As for the previous assay described, data
Glycolytic flux increases with increasing glu- are typically output in lifetime scale and
cose concentration and begins to saturate at then transposed into analyte scale using a
5 mM. Acidification increases further in predetermined calibration (Support Protocol
the presence of the Fo F1 ATPase inhibitor 5). This conversion can be done automatically
oligomycin, as cells become exclusively de- using customized data analysis tools available
pendent on glycolysis for cellular ATP and from plate reader manufacturers such as BMG
react by increasing glycolytic flux. Labtech and BioTek, or can be performed
The impact of drug treatment of 3D HepG2 manually.
cultures is presented in Figure 2.16.5C. Cells Sample MitoXpress-Intra based oxygena-
are cultured in the collagen-based RAFT sys- tion measurements of 3D HepG2 cultures are
tem, exposed to classical ETC modulators, and presented in Figure 2.16.6. Cells were pre-
measured immediately post-treatment, where- loaded with MitoXpress-Intra, grown in RAFT
upon mitochondrial impairment causes gly- culture, and subsequently measured across
colytic compensation that is detected as in- a range of oxygen concentrations using a
creased acidification. This is clearly apparent plate reader equipped with atmosphere con-
in Figure 2.16.5D, where H+ -based ECA is ex- trol. A significant disparity is evident between
pressed as a percentage of untreated values and metabolically competent (untreated) and in-
compared to parallel MitoXpress-Xtra-based hibited (antimycin-treated) cells, demonstrat- Assessment of Cell
Toxicity
oxygen consumption measurements. The ing the degree to which cell respiration
2.16.29
contributes to cell deoxygenation at reduced Hynes, J., Nadanaciva, S., Swiss, R., Carey, C., Kir-
ambient oxygen concentrations. While nor- wan, S., and Will, Y. 2013. A high-throughput
dual parameter assay for assessing drug-induced
moxia for hepatocytes is 6% O2 , these data
mitochondrial dysfunction provides additional
illustrate that maintaining cells at 6% is not predictivity over two established mitochondrial
sufficient to ensure this level pericellularly, toxicity assays. Toxicol. In Vitro 27:560-569.
where values can vary considerably depending doi: 10.1016/j.tiv.2012.11.002.
on respiration levels (Fig. 2.16.6B). Monitor- Lapidus, R.G. and Sokolove, P.M. 1993. Spermine
ing this parameter is therefore key to establish- inhibition of the permeability transition of iso-
ing a physiologically relevant cell model for lated rat liver mitochondria: An investigation of
mechanism. Arch. Biochem. Biophys. 306:246-
more predictive drug toxicity analyses. This is
253. doi: 10.1006/abbi.1993.1507.
underscored by the difference observed in gly-
Marroquin, L.D., Hynes, J., Dykens, J.A., Jamieson,
colytic flux for cells at 6% versus 21% O2 (Fig.
J.D., and Will, Y. 2007. Circumventing the Crab-
2.16.6C). These parameters that can be mul- tree effect: Replacing media glucose with galac-
tiplexed using a combination of MitoXpress- tose increases susceptibility of HepG2 cells to
Intra and pH-Xtra thereby enable an analysis mitochondrial toxicants. Toxicol. Sci. 97:539-
of the interplay between available oxygen and 547. doi: 10.1093/toxsci/kfm052.
glycolytic ATP generation. Nadanaciva, S. and Will, Y. 2009. Current con-
cepts in drug-induced mitochondrial toxicity.
Curr. Protoc. Toxicol. 40:2.15.1-2.15.9. doi:
Time Considerations 10.1002/0471140856.tx0215s40.
Basic Protocol 1 requires 1 hr to perform.
Basic Protocols 2, 3, and 4 each require 2-3 hr. National Research Council of the National
Academies (NRC). 2011. Guide for the Care
Support Protocol 1 requires 2-3 hr. Support and Use of Laboratory Animals, 8th ed. The
Protocol 2 requires 1 hr. Support Protocol 3 National Academies Press, Washington, D.C.
requires 30 min. Support Protocol 4 requires Ogurtsov, V.I., Hynes, J., Will, Y., and Papkovsky,
45 min. Support Protocol 5 requires 3 hr. D.B. 2008. Data analysis algorithm for high
throughput enzymatic oxygen consumption as-
says based on quenched-fluorescence detection.
Acknowledgments Sens. Actuators B Chem. 129:581-590. doi:
Some of the research presented here was 10.1016/j.snb.2007.09.004.
carried out as part of the HeCaTos project.
O’Riordan, T.C., Fitzgerald, K., Ponomarev, G.V.,
HeCaTos is funded by the European Union 7th Mackrill, J., Hynes, J., Taylor, C., and Pap-
Framework Programme (HEALTH-F4-2013- kovsky, D.B. 2007. Sensing intracellular oxy-
602156). gen using near-infrared phosphorescent probes
and live-cell fluorescence imaging. Am. J. Phys-
iol. Regul. Integr. Comp. Physiol. 292:R1613-
Literature Cited R1620. doi: 10.1152/ajpregu.00707.2006.
Chapple, S.J., Keeley, T.P., Mastronicola, D.,
Arno, M., Vizcay-Barrena, G., Fleck, R., Siow, Sung, H.J., Ma, W., Wang, P.Y, Hynes, J.,
R.C.M., and Mann, G.E. 2016. Bach1 differ- O’Riordan, T.C., Combs, C.A., McCoy, J.P. Jr.,
entially regulates distinct Nrf2-dependent genes Bunz, F., Kang, J.G., and Hwang, P.M. 2010. Mi-
in human venous and coronary artery endothe- tochondrial respiration protects against oxygen-
lial cells adapted to physiological oxygen lev- associated DNA damage. Nat. Commun. 1:5.
els. Free Radic. Biol. Med. 92:152-162. doi: doi: 10.1038/ncomms1003.
10.1016/j.freeradbiomed.2015.12.013. Toussaint, O., Weemaels, G., Debacq-Chainiaux,
Hynes, J., Marroquin, L.D., Ogurtsov, V.I., F., Scharffetter-Kochanek, K., and Wlaschek,
Christiansen, K.N., Stevens, G.J., Papkovsky, M. 2011. Artefactual effects of oxygen on cell
D.B., and Will, Y. 2006. Investigation of culture models of cellular senescence and stem
drug-induced mitochondrial toxicity using cell biology. J. Cell. Physiol. 226:315-321. doi:
fluorescence-based oxygen-sensitive probes. 10.1002/jcp.22416.
Toxicol. Sci. Off. J. Soc. Toxicol. 92:186-200. Wallace, K.B. 2008. Mitochondrial off targets of
doi: 10.1093/toxsci/kfj208. drug therapy. Trends Pharmacol. Sci. 29:361-
Hynes, J., O’Riordan, T.C., Zhdanov, A.V., Uray, 366. doi: 10.1016/j.tips.2008.04.001.
G., Will, Y., and Papkovsky, D.B. 2009. In vitro Will, Y., Hynes, J., Ogurtsov, V.I., and Pap-
analysis of cell metabolism using a long-decay kovsky, D.B. 2007. Analysis of mitochon-
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doi: 10.1016/j.ab.2009.04.016. 10.1038/nprot.2006.351.
Fluorescent Assay
for Mitochondrial
Function
2.16.30

Hynes 2016

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Fluorescence-Based Microplate Assays UNIT 2.

Wiley & Sons, Inc.

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Current Protocols in Toxicology 2.16.1-2.16.30, November 2016 2.16.1

MEASUREMENT OF OXYGEN CONSUMPTION IN ISOLATED

BASIC Measurement of Electron Transport Chain Activity

2. Reconstitute MitoXpress-Xtra probe in 1 ml measurement buffer 1, then dilute to

3. Warm mineral oil and diluted probe to 30°C.

For optimization of mitochondrial protein concentration

For basal respiration (State 2):

5a. Prepare a six-point dilution series of isolated mitochondria in measurement buffer 1

6a. Place a black 96-well plate on a plate heater equilibrated to 30°C.

Fluorescent Assay 100 µl diluted MitoXpress-Xtra probe

The final substrate concentration is 25 mM succinate or 12.5/12.5 mM glutamate/malate.

13. When optimizing the mitochondrial protein concentration:

Screens are typically performed at 0.5 mg/ml succinate or 1 mg/ml glutamate/malate

SUPPORT Isolation of Mitochondria from Rat Liver

Sterile dissection tools

Mitochondrial Protein Assay SUPPORT

2-ml microcentrifuge tubes

2. In 2-ml microcentrifuge tubes, prepare mitochondrial dilutions at 1:60, 1:80, and

SUPPORT Instrument Signal Check

Time-resolved fluorescence plate reader with temperature control and kinetic

3. In a 96-well plate on a prewarmed multiblock block heater, add 150 µl prewarmed

6. Insert microplate into the plate reader and commence reading.

MEASUREMENT OF METABOLIC PERTURBATION IN WHOLE CELLS

BASIC Cell-Based Measurement of Oxygen Consumption

4. Prepare the test compound dilution series in the appropriate solvent.

5. Add 16 ml growth medium to a 50-ml plastic tube and warm to 37°C.

7. Wash cells with fresh, prewarmed growth medium.

Cell Permeabilization SUPPORT

7. Wash cells with prewarmed respiration buffer.

MEASURING CELLULAR OXYGENATION AND ASSOCIATED

Measurement of Cellular Oxygenation Using Fluorescent Intracellular Oxygen BASIC

Prepare instrument and reagents

8. If measuring at defined O2 and CO2 concentrations, set the atmospheric control

Set up assay plate

For kinetic assay with compound treatment(s)

a. Add 1 µl of 150 μM antimycin A to designated wells to inhibit cellular oxygen

REAGENTS AND SOLUTIONS

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