Download Multiplexing of DELFIA® Cell Proliferation and DNA

Multiplexing of DELFIA® Cell Proliferation and DNA Fragmentation Assays with
Conventional Fluorometric Cell-Based Assays for Identification of Toxic Compounds
Sofia Vikström, Päivi Pulli, Pertti Hurskainen, Christel Gripenberg-Lerche
PerkinElmer Life and Analytical Sciences, Turku, Finland
Stimulate cells with
test compounds
Label cells for metabolism or
oxidative stress assay
Readout: Cell metabolism or
oxidative stress
DELFIA EuTDA
Membrane
TDA
integrity
BATDA
ATPlite
Metabolism
DELFIA Proliferation
DNA synthesis
ADP
General Cell death:
DNA synthesis È
ATPÈ
Label cells for cell
number control
Detection of incorporated
nucleotides is done using
components labeled with
different
lanthanide
chelates.
Measure the timeresolved fluorescence
Measure the
fluorescence
A label for total number of
cells
in
each
well
(SYTO24®) is also included
in the detection mix.
Readout: DNA
Fragmentation
Dye
Eu
TruPoint CaspaseCaspase-3
Apoptosis
Readout: Cell
Proliferation
Readout: Number
of cells/well
Apoptosis:
Caspase 3 acitivity Ç
DNA fragmentationÇ
5
oxidative stress
5
Toxic compounds may influence cells in many different ways,
through various mechanisms. All types of toxicity can not be
detected by one type of cell-based assay. Some of the potential
drug candidates may have a toxic effects on the cell, in which
case the toxicity can be detected when running a toxicity assay
simultaneously on the same sample. Time is also a critical
factor, some toxic effects are discovered after a short time of
treatment, while other require long incubations. Multiplexing
different types of cell-based toxicity assays gives more
information regarding the toxic properties of a compound. Since
the DELFIA® assays are based on time-resolved fluorometry,
they are readily combined with conventional fluorescence
assays. The lanthanide chelates are well suited for multiplexing
assays. The use of the lanthanide chelates will maximize
sensitivity and minimize signal spillover for the assays.
40000
160000
35000
140000
30000
120000
25000
100000
DNA
Fragmentation
20000
15000
Proliferation
80000
60000
4
0,5
3
1
0,3
0,1
0,03
0,01 0,003
0
Figure 6. CHO cells were cultured over night at 10000 cells/well in a 96-well plate. Staurosporine, 0 – 30µM, was added
and the cells were incubated for 6 hours before addition of the BrdU- and the alamarBlue labeling reagents. The cells were
incubated in the presence of alamarBlue and BrdU overnight. The absorbance was measured, and the cells were fixed
before detection of incorporated BrdU according to the DELFIA® Cell Proliferation kit insert.
3
1
0
Staurosporine
H2O2
Figure 5. Oxidative stress and DNA fragmentation in CHO cells. The
cells were loaded with CM-H2-DCFDA and treated with different
compounds inducing oxidative stress or apcptosis. The data is
expressed as signal from treated- compared to untreated cells.
10
8
Cell Proliferation, cell metabolism, and
DNA fragmentation assays
Four different assays are performed in the same well. The cell metabolism assay is an
absorbance measurement, the DELFIA® cell proliferation and the DNA fragmentation
assays are time-resolved fluorescence assays and the label to correct for unequal number
of cells/well is a conventional fluorescence label.
Cell metabolism
(alamarBlue)
amount of cells (SYTO24)
5
0
100
1000
10000
num ber of cells/w ell
6
5
9
4
Conclusions
3
Cell-based DELFIA® assays can be multiplexed and combined with assays and
applications using conventional fluorescence measurements.
2
20000
1
0
0
CTRL
DNA fragmentation
7
6
Figure 7. CHO cells were plated at an increasing concentration. The cells were treated with 10µM STS for 6 hours. The
data shows that the cell metabolism (alamarBlue) and the cell proliferation are not significantly affected by the STS
treatment, while the DNA Fragmentation increases. Some of the cells are washed away due to the STS treatment, the
control for equal amount of cells/ well is used to correct for this.
7
40000
Figure 3. The data shows proliferation and DNA fragmentation in CHO cells,
10000 cells/well, grown overnight. The cells were treated with 10µM staurosporine
(STS), for 6 hours to induce apoptosis. Apoptotic cells were compared to untreated
cells. The data is expressed both as Samarium counts for the DNA fragmentation
assay, and as Europium counts for the proliferation assay.
Proliferation (BrdU)
9
8
2
1
Both assays
8
10
4
3
One assay
9
5000
ST S
0,6
10
[Staurosporine (µM)]
2
10000
0
0,6
30
DNA Fragmentation
200000
180000
alamarBlue grow th
assay
0,7
300000
0
The DELFIA® Cell Proliferation assay is based on the incorporation of BrdU into newly synthesized DNA strands of proliferating cells. Incorporated BrdU is
detected using a labeled antibody. The DNA Fragmentation assay is a cell-based assay performed in 96-well micro plate format for quantitative detection of
apoptosis. The labeling of the 3’-hydroxyl ends of DNA fragments provides a measure of cells undergoing apoptosis. Incorporated, labeled nucleotides can
be detected by labeled streptavidin. We have combined this two assays, Europium-labeled anti-BrdU antibody for the proliferation assay, and Samariumlabeled streptavidin for the DNA fragmentation assay.
45000
0,7
400000
DELFIA Cell
Proliferation
100000
Cell Proliferation & DNA fragmentation assays
Sam ariu m co u n ts
Multiplexing of cell-based assays
007242_06
6
All
the
assays
are
measured
using
the
multilabel plate readers
VICTOR™ and EnVision™.
50000
0,8
500000
DNA
Fragmentation
Apoptosis
Figure 1. Non-radioactive cytotoxicity assays are used primarily in secondary screening in the drug
discovery process. There is a trend, for early cell toxicity testing to identify compounds with toxic properties
before lead optimization. These kinds of assays are also used in basic research, to study cell proliferation,
apoptosis, etc. in for instance cancer research and immunology. The cell-based assays of the ToxSuite
provide extensive information about the cytotoxic effects of compounds.
3
600000
S/B
ATP
Label with antibodies for
time-resolved fluorometry
0,8
700000
200000
The TUNEL reaction for
quantification
of
DNA
fragmentation is performed
directly in the wells on the
fixed cells.
Membrane damage:
Intracellular marker
release Ç
Cell Proliferation & alamarBlue assays
Cell proliferation by incorporation of BrdU, and detection of metabolic activity in cells using
the growth indicator alamarBlue. The BrdU- and the alamarBlue labeling reagents were
added simultaneously, The homogenous alamarBlue assay is performed first and then the
cells are fixed for the end-point DELFIA® Cell Proliferation assay.
7
S /B
Fixation of cells
TUNEL reaction for DNA
Fragmentation assay
TDA
The labeling of the cells for
the proliferation assay, for
the oxidative stress assay,
and for the metabolism
assay is done with live
cells, without removing the
culture media or the test
compound.
After
performing
the
measurement for the cell
metabolism assay, the
culture media is removed,
and the cells are fixed.
Overview of the ToxSuite Products
Proliferation:
ATPÇ
DNA synthesis Ç
The cells are loaded with CM-H2-DCFDA, treated with test
compounds, and the oxidative stress is monitored in live-cells. CMH2-DCFDA is a cell-permeable indicator of reactive oxygen
species. The indicator remains non-fluorescent until removal of the
acetate groups by intracellular esterases and until oxidation occurs
within the cell. After live-cell, kinetic measurement of oxidative
stress the cells can be fixed and the cell-based assay for DNA
fragmentation performed.
All
the
assays
are
performed simultaneously
on the same samples, and
in the same wells. In
addition to increasing the
amount of data gained form
each sample, this also
saves reagents, plates, and
test compounds.
7
Absorbance
Grow cells in
96 well plates
Label cells with BrdU for
proliferation assay
Oxidative stress & DNA
fragmentation
Figure 2. Assay flow chart
for multiplexing.
S/B
2
6
Assay Flow Chart
Eu counts
4
Introduction
We have developed sensitive assays for Apoptosis, cell metabolism
and cell proliferation monitoring these events. These non-radioactive
assays can be used in secondary screening in the drug discovery
process, for early cell toxicity testing, to identify compounds with toxic
properties. Combining different assays provide more information
regarding the cytotoxic properties of compounds. The DNA
Fragmentation assay is a simple, cell-based TUNEL assay for
quantitative detection of apoptosis, performed in microplate format.
The DELFIA® Cell Proliferation assay measures the effect of growth
regulatory substances. These assays can also be combined with one
another, or combined with other conventional fluorometric assays or
applications. We have set up cell-based assays where the DELFIA®
detection technology is combined with conventional fluorometry, and
where both assay technologies are performed on the same sample.
Eu ro p iu m co u n ts
1
Multiplexing of DELFIA® assays saves time as well as reagents and more data is
gained from one sample.
D N A F r ag ment at io n
Pr o lif erat io n
Figure 4. Proliferation and DNA fragmentation in CHO cells treated with
STS. The two assays do not significantly affect each other when
performed simultaneously in the same sample wells. The data is
expressed as signal to background for both the proliferation- and the
DNA fragmentation assays when performed either alone or together.
All assays can be performed on the same plate and read on the same timeresolved fluorescence reader.
When performing cell-based assays where cells are going through various
treatments, it is important to include a control for equal number of cells in each
well. We have used a nucleic acid stain for this. The label is not interfering with
the time-resolved fluorescence.
PerkinElmer Life and Analytical Sciences, 710 Bridgeport Avenue, Shelton, CT USA (800) 762-4000 (+1) 203 925-4602 www.perkinelmer.com