Application note:Layout 1 14/1/10 22:28 Page 51
Application Note
Determine predictive mechanisms of toxicity
using a single-well multiplexed assay
By Sarah Shultz, dead-cell protease activity. Dead-cell pro-
cell-permeant
Promega Corporation teases are released from cells that have
GF-AFC
substrate
lost membrane integrity. The live- and
inactive
live-cell
dead-cell proteases produce different fluo-
GF-AFC live-cell
protease
substrate protease
noncell-permeant
bis-AAF-R110 active
M
ultiplexing can be defined as the rescent products with distinct excitation
substrate dead-cell
protease
use of compatible assays to meas- and emission spectra allowing them to be
AFC
ure multiple parameters in a sin- detected simultaneously
1
using a fluores-
R110
gle well. One significant advantage, particu- cent plate reader.
Viable Cell Dead Cell
5847MA
larly in secondary screening, is generating Caspase activity is measured using a
Figure 1: Viability and cytotoxicity are measured
more biologically relevant data. Information luminogenic substrate in a subsequent step
as fluorescent readouts in the first step of the
about cell health, toxicity and apoptosis in the assay. The luminescent signal is pro-
ApoTox-Glo™ Assay. A second step measures
events from the same population of cells pro- portional to the amount of caspase activity caspase activity as a bioluminescent readout
vides a broader picture of what is occurring present. Assay configurations for multiwell
inside cells and is particularly well suited for in plate formats are provided in Table 1.
Viability EC
50
= 13.6µM
Cytotoxicity EC
50
= 21.6µM
vitro toxicology applications.
Apoptosis
50,000 800,000
)
Performance of the assay
t
y
ci
)
R
LU
40,000
600,000
t
o
xi
R
FU
The assay simplifies multiplexing Cell viability, cytotoxicity, and caspase-3/7
y
t
o
30,000
C 400,000 in
escence (
The ApoTox-Glo™ Triplex Assay is activity were assessed using the assay in
escence (
20,000
l
i
t
y or
uor 200,000
s
Lum
designed to measure cell viability, cytotox- K562, Jurkat, L929, and HepG2 cell lines
Fl 10,000 osi
V
i
abi
0 0 A
popt
icity, and caspase activation in each well treated with mechanistically different cyto-
–7 –6 –5 –4
Log
10
[ionomycin], M
8224MA
in common multiwell plates. The assay toxic compounds (ionomycin, staurosporine,
measures two protease biomarker activi- bortezomib and SAHA). The cytotoxic pro- Figure 2: Representative data of the ApoTox-
ties, a live-cell marker (cell viability) and a files generated using the assay demonstrate
Glo™ Triplex Assay. K562 cells in suspension
dead-cell marker (cytotoxicity), as illus- the expected trends for the effect of each
were treated for four hours with ionomycin in
96-well format resulting in a dose-dependent
trated in Figure 1. The live-cell protease compound on the cells (Figure 2 and
decrease in viability and increase in cytotoxicity
activity is restricted to intact viable cells http://www.promega.com/pubs/tpub_011.ht
with no caspase-3/7 activation, which is
and is measured using a fluorogenic, cell- m). In vitro cytotoxicity is dependent upon consistent with primary necrosis
permeant substrate, which enters intact dosage of compound and time of exposure.
cells and is cleaved by the live-cell pro- The kinetics of measurable cytotoxic bio- important to characterise new compounds
tease. A second, distinct fluorogenic cell- markers can vary widely between individual using multiple exposure periods.
impermeant substrate is used to measure compounds and treatments. Therefore, it is
Conclusion
Multiplexing with the Promega ApoTox-
96-WELL 384-WELL 1536-WELL Glo™ Triplex Assay helps to provide
researchers with information about cell
Recommended cell 10,000-20,000 5,000-10,000 1,000-5,000
health, toxicity and apoptosis events,
density (cells/well)
while minimising the assay workflow time.
Add cells + cmpds in 100µl 20µl 4µl
The assay is easily configured to work
culture medium across multiwell plate formats and serves
Treat cells at 37°C in 5% CO
2
for the desired amount of time (ie, 4-48 hrs)
as an especially useful tool to better under-
Add viability/ 20µl 5µl 1.25µl
stand and predict the mechanism of cellu-
cytotoxicity reagent
Incubate at 37°C for 30 minutes. Measure fluorescence lar cytotoxicity.
Add Caspase-Glo® 3/7 100µl 25µl 4µl
Reagent
Incubate at room temperature for 30 minutes. Measure luminescence
Reference
1 Niles, AL et al (2007). A homogeneous assay
Table 1: ApoTox-Glo™ Triplex Assay protocol incorporates a simple sequential ‘add-mix-read’ to measure live and dead cells in the same
format. The volumes of each assay component can be scaled to meet varying researchers' throughput sample by detecting different protease markers.
needs and is highly amenable to automation Anal. Biochem 366, 197–206.
Drug Discovery World Winter 2009/10 51
Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88