Application Note

Screening for Mitochondrial

Biogenesis and Toxicity
A sensitive and robust cell-based screening assay

Research Area

Obesity, Diabetes & Metabolic

Disorders
in vitro toxicity
Neurodegeneration

Mitochondrial damage compromises ATP production and consequentially cellular viability. Given the
central role that mitochondria play in regulating cellular function, drugs that undermine mitochondrial
function frequently elicit toxic effects. In contrast, mitochondrial biogenesis increases cell vitality and is
being pursued as a strategy to combat age-related diseases. Thus, there is growing interest in identifying agents that induce biogenesis.

Aging

Currently there are no real-time screening assays that assess multiple parameters of mitochondrial func-

Cancer

tion. Methods for determining mitochondrial function by measuring cellular respiration have relied largely
on Clark electrode chambers that lack the throughput needed for screening. In addition, many cultured

Assay type

cell lines exhibit altered mitochondrial physiology that does not correlate well to

Mitochondrial function:

in vivo mitochondrial biogenesis and toxicity.

mitochondrial biogenesis

The XF96 Extracellular Flux Analyzer addresses the need for higher throughput respirometric measure-

and toxicity

ments. To assess its utility in screening, primary cultures of renal proximal tubular cells (RPTCs) from
rabbit were optimized for the XF platform and tested with well characterized nephrotoxicants and mito-

Keywords

mitochondrial respiration
mitochondrial biogenesis
toxicity
renal proximal tubular cells
compound screening

chondrial biogenesis activators.

Beeson et al [1] adapted primary cultures of RPTC to exhibit in vivo levels of aerobic metabolism, to be
non-glycolytic, and to retain high levels of differentiated functions. The XF96 Analyzer was used to measure mitochondrial respiration of the RPTC in real time. Changes in carbonylcyanide-4-(trifluoromethoxy)
-phenylhydrazone (FCCP)-uncoupled respiration, a measure of electron transport chain (ETC) integrity
and a sensitive measure of mitochondrial functional capacity, were used to quantify mitochondrial toxicity and biogenesis as described by Beeson [1]. The nephrotoxicants cisplatin, HgCl2 and gentamicin

FCCP-uncoupled rates expressed as

the ratios of treated to control values for
each of the wells are shown. The different
colored data clusters discriminate plates
and horizontal lines indicate 1 and 3
standard deviations above and below
the mean. Agents that produce ratios
with standard deviations 1 are potential
biogenesis inducers and those with
standard deviations -1 are potential
toxicants.

2.4
STDV=3
STDV=1
MEAN
STDV=-1
STDV=-3

2.2
Treated FCCP Uncoupled Rate/Vehicle Control

Figure 1 | Representative data

for a primary screening assay
of mitochondrial biogenesis
and toxicity.

2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6

200

400

600
Number of Compounds

800

1000

1200

Screening for Mitochondrial Biogenesis and Toxicity

induced statistically significant decreases in the FCCP-uncoupled rates prior to decreases in basal respiration and cell death. Conversely, drugs known to induce mitochondrial biogenesis such as
1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin
produced statistically significant increases in FCCP-uncoupled respiration rates [1]. Decreases or
increases in FCCP-uncoupled respiration rates can function as measures of mitochondrial toxicity or
biogenesis, respectively.
To validate the RPTC platform as a primary screening assay, the 1280-compound LOPAC library was
screened. Cells were treated with library compounds at 5 M using one compound/well in duplicate.
After 24 h treatment, the basal and uncoupled (maximal) oxygen consumption rates [OCR] were measured with the XF96 Analyzer.
Figure 1 shows representative data from a single pass of the primary screening assay of the LOPAC library.
End-point measurements of FCCP-uncoupled rates expressed as the ratios of treated wells to vehicle control showed that most compounds had little effect, demonstrating good selectivity for active compounds.
Compounds known to promote mitochondrial biogenesis showed increases in the FCCP-uncoupled rates
on the order of 2050% above controls (>1 standard deviation from the mean.) Conversely, compounds

The assay takes advantage of

both the high throughput and
precision oxygen consumption
rate measurements afforded by
the XF96 platform.

known to elicit toxicity display decreases in the FCCP-uncoupled rates on the order of 2050% below
controls. In this screen the compounds that elicit increases or decreases above or below one standard
deviation are the most likely candidates for biogenesis inducers or toxicants, respectively. Positive controls
for both classes of compounds consistently lie within the same ranges. The merger of the RPTC model
and the Seahorse XF96 Analyzer results in an primary screening assay to simultaneously measure mito-

Given that impairment of mitochondrial function is implicated

chondrial biogenesis and toxicity including nephrotoxic potential.

in metabolic disorders such

Discussion

as diabetes, neurodegenera-

The validity of this screen is due to the robust mitochondrial function of the RPTC and the sensitivity of

tive diseases, heart failure, and

the FCCP-uncoupled respiration rates to detect dysfunction or enhanced function of mitochondria. The
assay format takes advantage of the high throughput afforded by the XF96 platform and the high precision of its oxygen consumption rate measurements [OCR]. Combining the primary RPTC model with the

aging in general, this assay will

enhance the development of

XF96 platform produces the first high throughput phenotypic assay of mitochondrial function that can be
used as a primary screen to identify stimulators of mitochondrial biogenesis or compounds with potential
toxicity. It can also be employed as a secondary or tertiary screen to identify and confirm mitochondrial
biogenesis inducers or mitochondrial toxicants. Given that impairment of mitochondrial function is implicated in metabolic disorders such as diabetes, neurodegenerative diseases, heart failure, and aging in
general, this assay will enhance the development of new therapeutic agents.
In another study, the XF96 Analyzer was evaluated as an assay platform to screen for drug-induced
mitochondrial impairment using both cell lines and primary cultures (manuscript in preparation). In
addition to confirming drugs with known adverse effects on mitochondrial function and glycolysis, the
authors tested several drugs including Tolcapone and Entacapone, used in the treatment of Parkinsons
disease; Nilutamide and Flutamide, anti-androgens given in the treatment of prostate cancer; and the
anti-diabetic drugs Troglitazone, Ciglitazone and Pioglitazone. In these experiments, it was shown that
the XF96 Analyzer had intra- and inter-assay variations of less than 15%, demonstrating the broad applicability of the XF96 across different cellular screening platforms.
Gohil et al [2] used the ratio of OCR to ECAR, a measure of glycolysis, to create an aerobic quotient.
They used this assay as a secondary screen to identify drugs that shift energy metabolism from mitochondrial respiration to glycolysis [extracellular acidification rate]. This very unique and informative assay
is capable of screening for compounds that shift tumor cells towards a more treatable aerobic metabolic
state or ones that shift cardiomyocytes or neurons to a more glycolytic state which may be more cardioprotective or neuroprotective.

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new therapeutic agents.

Screening for Mitochondrial Biogenesis and Toxicity

Materials and Methods

Cells: RPTC were isolated using the iron oxide perfusion method as described previously [3]. The
resulting proximal tubules were plated on 100-mm tissue culture plastic petri dishes. Plates were constantly swirled on an orbital shaker at 80 rpm. After 3 days, RPTC were removed with trypsin, and
plated into the wells of XF96 cell culture microplates at 18,000 cells/well. The plates were shaken on an
orbital shaker at 80 rpm for 4 days, treated, and assayed 24 h later. The medium is a 50:50 mixture of
Dulbeccos modified Eagles essential medium and Hams F12 nutrient mix without phenol red, supplemented with 15 mM NaHCO3, 0.2 mM glycine and 6 mM sodium lactate. The medium is adjusted to pH
7.4 while gassing with 95% O2-5% CO2 and diluted to 295 mosmol/kg H2O before filter sterilization. The
medium is then supplemented with human transferrin (5 g/mL), selenium (5 ng/mL), hydrocortisone (50
nM), and bovine insulin (10 nM).
The LOPAC library was obtained from Sigma-Aldrich. Stocks are 10 mM in DMSO.
XF Analysis
XF analyses were performed in the XF96 Extracellular Flux Analyzer (Seahorse Bioscience,) a fully integrated, 96 well format instrument that measures the uptake and excretion of metabolic end products in
real time. Oxygen consumption rates [OCR] were measured using XF assay kits. Assay kits each contain
a disposable sensor cartridge, embedded with 96 immobilized (solid-state) dual-fluorescent biosensors
(oxygen and pH.) Each sensor cartridge is also equipped with drug injection chambers for delivering
testing agents into wells during an assay. OCR is expressed as pmoles/min. ECAR was not utilized in
this protocol.
As illustrated in Figure 2, duplicate wells were treated with 5 M compound in 200 L media supplemented with 10 mM HEPES. Each plate also had vehicle control, no treatment, and SRT1720 positive
control wells, in duplicate. After 24 hours the plates were analyzed on an XF96 Analyzer without
exchanging the culture medium. During the experiment five basal OCR were measured with a 4 minute
mix and 1 minute measurement cycle. Each well was injected with 0.5 M final concentration of FCCP in
25 L media followed by three OCR measurements using a 2 minute mix and 1 minute measurement
cycle. The average of the three uncoupled rates for each treated well are divided by the average of the
three uncoupled rates for the vehicle control wells to give the ratios illustrated in Figure 1. Quality control
assessments include comparisons between basal and uncoupled rates of vehicle control versus no
treatment control wells, and comparisons of the control well uncoupled rates as a ratio of the averaged
basal rates; the latter ratios range from 1.53.0 for acceptable assays.

Figure 2 | Flow Chart of XF Assay

7 Days Prior to Day of Assay

Prior to Day of Assay

Renal proximal tubular cells

(RPTCs) were isolated from
female New Zealand white
rabbits and cultured 3 days in
shaking 100 mm Petri dishes.

Prepare FCCP Stock

Seed RPTCs at
18,000 cells/well and
culture for 4 days
while swirling

Day of Assay

Dilute FCCP

Load Cartridge
& Calibrate
15 minutes

Run Experiment
1.5 hours
Treat duplicate wells with
5 uM compound from the
LOPAC library

Analyze Data
1 hour

No Medium Change

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References
1. Beeson CC, Beeson GC, Schnellmann RG. A high-throughput respirometric assay for mitochondrial biogenesis and toxicity. Anal Biochem. 2010 Sep 1; 404(1):75-81. Epub 2010 May 15.
2. Gohil VM et al. Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial
respiration to glycolysis. Nature Biotech. 2010;28(3):249-57.
3. Nowak G, Schnellmann RG. L-ascorbic acid regulates growth and metabolism of renal cells: improvements
in cell culture. Am J Physiol. 1996;271(6 Pt 1):C2072-80.

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