2012 Promega catalogue

Life
Science
Catalog
2012
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Cell Signaling
Mitochondrial Function Assay
46
Mitochondrial Toxicity Assay
Product Size Cat.# Price ($)
Mitochondrial ToxGlo Assay 10 ml G8000 286.00
For Research Use Only. Not for Use in Diagnostic Procedures.
100 ml G8001 1678.00
Description: The Mitochondrial ToxGlo Assay is a cell-based assay method
that employs sequential addition, multiplexed assay chemistry for predicting
potential mitochondrial dysfunction as a result of xenobiotic exposure. The
assay is based on the differential measurement of biomarkers associated
with changes in cell membrane integrity and cellular ATP levels relative to
vehicle-treated control cells during short exposure periods. Cell membrane
integrity is first assessed by measuring the presence or abscence of a distinct
protease activity associated with necrosis using a fluorogenic peptide substrate
(bis-AAF-R110) to measure “dead cell protease activity”. The bis-AAF-R110
Substrate cannot cross the intact membrane of live cells and therefore gives no
signal with viable cells. Next, ATP is measured by adding an ATP detection reagent,
resulting in cell lysis and generation of a luminescent signal proportional
to the amount of ATP present. The two sets of data can be combined to produce
profiles representative of mitochondrial dysfunction or non-mitochondrial
related cytotoxic mechanisms.
Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation)
and nonmitochondrial (glycolysis) methods. To achieve optimal mitochondrial
responsiveness, replacing glucose-supplemented medium with galactosecontaining
medium may increase cellular oxygen consumption and augment
mitochondrial susceptibility to mitotoxicants.
A.
150
ATP
Cytotoxicity
B.
Percent Vehicle Control
100
50
0
100
50
0
–7 –6 –5
Log [imipramine], M
C. D.
150
Percent Vehicle Control
–7 –6
Log [CCCP], M
–5
Features:
• Distinguish Primary Mitochondrial Dysfunction from Secondary
Cytotoxic Events: Cell-based, multiplexed method measures ATP in
conjunction with a membrane integrity biomarker to distinguish primary
mitochondrial dysfunction from secondary cytotoxic events.
• Predictive for Mitochondrial Toxicities: Produces profiles that are consistent
with mitochondrial toxicity and discernible from other nonmitotoxic
mechanisms of cell death.
• Easy to Implement: The assay uses a simple sequential “add-mix-read”
format to assess toxicity in the sample well.
• Fast: Quickly assess potential mitochondrial liabilities in under an hour.
• Cost-Effective: Assays are performed directly in cell culture plates using
standard multimode detection instrumentation.
• Flexible and Easily Automated: The volume of reagent addition can be
scaled to meet throughput needs; the assay is amenable to automation in
96- and 384-well plates.
Storage Conditions: Store the Mitochondrial Tox-Glo Assay components
at –20°C.
Protocol Part#
Mitochondrial ToxGlo Assay Technical Manual TM357
Percent Vehicle Control
Percent Vehicle Control
250
200
150
100
50
0
600
500
400
300
200
100
–6 –5
Log [digitonin], g/L
–4
0
–7 –6 –5 –4
Log [antimycin], g/L
Representative profiles of mitochondrial toxicity with the Mitochondrial ToxGlo Assay. K562 cells were plated at 10,000 cells/well in 96-well
plates and treated with serial dilutions of compounds resuspended in glucose-free (galactose-supplemented) RPMI 1640 medium for 2 hours.
Panel A shows no changes in ATP or membrane integrity (MI), which indicates that the compound is not a mitochondrial toxin. Panel B. The reduction in ATP
with commensurate MI changes indicate that the compound is not a mitochondrial toxin; instead primary necrosis is taking place. Panel C. The reduction in
ATP with no changes in MI indicates that the compound is a mitochondrial toxin. Panel D. The reduction in ATP with discordant changes in MI indicate that the
compound is a mitochondrial toxin.
For complete and up-to-date product information visit: www.promega.com/catalog
9955MA