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2012
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Cell Signaling
ApoTox-Glo Triplex Assay
Product Size Cat.# Price ($)
ApoTox-Glo Triplex Assay 10 ml G6320 828.00
For Laboratory Use.
5 × 10 ml G6321 Pls. Enq.
Description: The ApoTox-Glo Triplex Assay combines three chemistries to
easily assess viability, cytotoxicity and apoptosis events in the same assay well.
Viability and cytotoxicity are determined by measuring two differential protease
biomarkers simultaneously with the addition of a single nonlytic reagent
containing two peptide substrates. The live-cell protease activity is restricted
to intact viable cells and is measured using a fluorogenic, cell-permeant
peptide substrate (GF-AFC Substrate). The substrate enters intact cells, where
it is cleaved to generate a fluorescent signal proportional to the number of
living cells. This live-cell protease activity marker becomes inactive upon loss
of membrane integrity and leakage into the culture medium. A second, cellimpermeant,
fluorogenic peptide substrate (bis-AAF-R110 Substrate) is used
simultaneously to measure dead-cell protease activity that has been released
from cells that have lost membrane integrity. This results in ratiometric,
inversely correlated measures of cell viability and cytotoxicity. The ratio of viable
cells to dead cells is independent of cell number and, therefore, can be used to
normalize data. A second reagent containing luminogenic DEVD-peptide substrate
for caspase-3/7 and Ultra-Glo Recombinant Thermostable Luciferase
is added. Caspase-3/7 cleavage of the substrate releases luciferin, which is a
substrate for luciferase and generates light. The light output, measured with
a luminometer, correlates with caspase-3/7 activation as a key indicator of
apoptosis.
Features:
• Measure Viability, Cytotoxicity and Apoptosis in the Same Sample
Well: Determine mechanism of cell death for cells in the same sample
well.
• Easily Implement: Assay follows a simple sequential “add-mix-measure”
format.
• Normalize Data with a Built-In Control: The ratio of the number of live
cells/number of dead cells is independent of cell number and normalizes
data. This normalization makes results more comparable well-to-well,
plate-to-plate and day-to-day.
• Flexible and Easily Automated: The volumes of each assay component
can be scaled to meet throughput needs and is amenable to automation in
96- and 384-well plates.
• Improves Efficiency and Saves on Lab Budget: Reduces cell culture
and labor costs by performing three assays in a single well.
Storage Conditions: Store all components at –20°C protected from light.
Protocol
ApoTox-Glo Triplex Assay Technical Manual
Part#
TM322
ApoLive-Glo Multiplex Assay
Product Size Cat.# Price ($)
ApoLive-Glo Multiplex Assay 10 ml G6410 775.00
5 × 10 ml G6411 Pls. Enq.
For Laboratory Use.
Description: The ApoLive-Glo Multiplex Assay measures both the number
of viable cells as a marker of cytotoxicity and caspase activation as a marker
of apoptosis within a single assay well to determine the mechanism of cell
death. The first part of the assay measures the activity of a protease marker of
cell viability. The live-cell protease activity is restricted to intact viable cells and
is measured using a fluorogenic, cell-permeant, peptide substrate (glycylphenylalanyl-amino
fluorocoumarin; GF-AFC). The substrate enters intact cells,
where it is cleaved by the live-cell protease activity to generate a fluorescent
signal proportional to the number of living cells. This live-cell protease becomes
inactive upon loss of cell membrane integrity and leakage into the surrounding
culture medium. The second part of the assay uses the Caspase-Glo ® Assay
technology to detect caspase activation, a key biomarker of apoptosis. The
Caspase-Glo ® Assay provides a luminogenic caspase-3/7 substrate, which
contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase
activity, luciferase activity and cell lysis. Adding the Caspase-Glo ® 3/7 Reagent
in an ‘add-mix-measure’ format results in cell lysis, followed by caspase
cleavage of the substrate and generation of a ‘glow-type’ luminescent signal
produced by luciferase. Luminescence is proportional to the amount of caspase
activity present.
Features:
• Measure Viability and Apoptosis in the Same Well: Accurately
determine the mechanism of cell death in less time with less sample.
• Easy to Implement: The assay uses a simple ‘add-mix-measure’ format.
• Normalize Caspase Data with Viability Control: The ratio of caspase
activity to viable cells is useful for determining the extent of caspase
activation and for normalizing cell numbers.
• Flexible and Easily Automated: The volumes of each assay component
can be scaled to meet throughput needs, and the assay is amenable to
automation in 96- and 384-well plates.
• Reveal cell death even if the window of caspase activity is missed.
• Multiplex with Other Assays: The nonlytic nature of the first step of the
assay allows further multiplexing with spectrally distinct fluorescent assay
chemistries.
Storage Conditions: Store all components at –20°C protected from light.
Protocol
ApoLive-Glo Multiplex Assay Technical Manual
Part#
TM325
Section
Contents
Table of
Contents
26
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