April 1999 Issue C15
CYTOSTAR-T SCINTILLATING MICROPLATE-BASED ASSAY FOR THE
MEASUREMENT OF APOPTOSIS
A homogenous radiochemical assay for measuring the binding of radiolabelled annexin V to
apoptotic cells has been developed. The assay uses Cytostar-T
 scintillating microplates,
and can be adapted for use with both adherent and suspension cells.
During early apoptosis, phosphatidylserine (PS) translocates to the outer surface of the plasma
membrane, where its surface expression makes it a potential marker for apoptosis(1). This
phenomenon is the basis for apoptosis assays which utilize the Ca2+ dependent high affinity binding of
annexin V to PS(1). A homogenous radiochemical assay for measuring the binding of annexin V to
apoptotic cells has been developed using Cytostar-T scintillating microplates (Figure 1). The assay
utilizes annexin V-biotin and [35S]streptavidin, and is compared to a method using directly labelled
[125I]annexin V. Flow cytometry was used to validate the radiochemical assays(1).
Annexin V and radiolabel
added in Ca2+ binding buffer
KEY
[35S]streptavidin
Annexin V-biotin
Phosphatidylserine
Cellular binding
of radiolabel
Decay of free radiolabel
in growth medium is too
distant to produce signal
Opaque well walls
Scintillating
base plate
Optical crosstalk mask
Light emitted isotropically
Figure 1. Cross-section from a well of a 96-well Cytostar-T scintillating microplate illustrating the
principle of the apoptosis assay.
To evaluate the assay for the measurement of apoptosis, two members of the TNF cytokine family
were examined, lymphotoxin (LT) which binds to TNFR1 and 2(2) and Apo2 Ligand (Apo-2L) which
binds DR4 and 5(3). Both are potent inducers of apoptosis in a wide variety of tumour cell types.
Using the 96-well Cytostar-T scintillating microplate-based assay, dose dependent binding of annexin
V-biotin and [35S]streptavidin was observed for L-M cells treated with LT (Figure 2). The assay was
homogenous in that no separation of bound and free annexin V was required. The response was
associated with an equivalent decrease in cell viability as measured by crystal violet staining, and was
consistent with the flow cytometry data (Figure 3).
0.5
35
0.4
6000
0.3
4000
0.2
2000
0
OD540nm
[ S]cpm
8000
0.1
0
1
10
0.0
100
Lymphotoxin (pg/ml)
Figure 2. Increased binding of annexin V-biotin and [35S]streptavidin to LT-treated L-M cells (■)
correlates with a decrease in cell viability, measured by crystal violet staining ( ). Cells were seeded
in a Cytostar-T scintillating microplate at 4 x 10-4 cells/well in phenol red free DMEM:Ham’s F12
(50:50), containing 10% FBS. After O/N incubation with 1µg/ml actinomycin D, 0.1ml fresh medium
containing 0.5-62pg LT/ml was added per well, followed by a second O/N incubation(4). Wells then
received 0.2µg annexin V-biotin in 50µl supplied 2x Ca2+ 'binding buffer' (containing 2.5% w/v BSA),
with incubation for 20 mins at RT. 50µl binding buffer was then added per well containing 0.5µCi
[35S]streptavidin (Amersham Biosciences, SJ436) with incubation for 2 hours at RT with gentle
shaking. The microplate was centrifuged for 8 mins at 1100rpm and counted on a Wallac 1450
MicroBeta. Media was removed, the wells washed once with PBS, and the cells stained with crystal
violet (0.5% in methanol) and dried. OD540nm was measured on an SLT 340 ATC plate reader. All
results are triplicate data points ± SD, and NSB background counts are subtracted.
(a)
% apoptosis
100
75
50
25
0
0
1
10
Lymphotoxin (pg/ml)
100
(b)
% apoptosis
80
60
40
20
0
0
0.001
0.01
0.1
1
10
ngApo-2L/ml
Figure 3. Quantitation of apoptosis with flow cytometry by measuring the binding of (■) annexin VFITC, or (†) annexin V-biotin and streptavidin-FITC, for (a) L-M cells treated with LT, and (b) H460
cells treated with Apo-2L. The labelled annexins were from NeXins Research BV, the Netherlands, and
the streptavidin-FITC was from DAKO Corporation, CA. Full methodology is given in Reference 5.
As shown in Figure 4, equivalent results were obtained using annexin V-biotin/[35S]streptavidin or
[125I]annexin V. However, the use of the [125I]annexin V required aspiration of the medium from the
wells before reading the microplate, to reduce the significant cross-talk (approximately 10%) which
was observed.
4000
6000
2000
2000
0
I]cpm
3000
4000
125
35
5000
[
[ S]cpm
8000
1000
0
1
10
0
100
pg Lymphotoxin/ml
Figure 4. Comparison of Cytostar-T apoptosis assay using annexin V-biotin and [35S]streptavidin (†)
and [125I]annexin V („) for L-M cells treated with lymphotoxin. The 'indirect' method using
biotinylated annexin V was performed as in Figure 2. For the 'direct' method, conditions were identical
except that 0.5µCi [125I]annexin V (Amersham Biosciences, IMQ20035, custom preparation) was added
in 50µl 3x Ca binding buffer per well, followed by RT incubation for 20 mins and centrifugation.
Medium was then aspirated from the wells before the microplate was counted.
In order to determine that the Cytostar-T scintillating microplate assay signal was not influenced by
the presence of necrotic cells, necrosis was induced by treatment with repeated freeze/thawing. No
signal was measurable after this treatment (not shown). The utility of the assay with a wide range of
cell lines, including suspension cells, is shown in Table 1.
Cell Name
Cell Type
Growth
ED50(µ
µg/ml)
A549
lung carcinoma
adherent
0.311±0.023
NCI-H460
lung carcinoma, large cell
adherent
0.017±0.004
ME-180
cervical carcinoma
adherent
0.233±0.025
SK-MES-1
lung carcinoma, squamous
adherent
0.066±0.009
SK-BR-3
breast adenocarcinoma
adherent
0.048±0.003
BT474
breast ductal carcinoma
adherent
0.947±0.031
HCT 146
colon carcinoma
adherent
0.112±0.017
HCT 116
colon carcinoma
suspension
0.077±0.012
9D
B cell
suspension
0.033±0.009
HUVEC
umbilical vein endothelial
adherent
2.560±0.145
HMEC
mammary epithelial
adherent
negative
WI-38
lung fibroblast
adherent
negative
Table 1. Detection of apoptosis in multiple cell lines treated with Apo-2L, using Cytostar-T scintillating
microplates to measure the binding of annexin V-biotin and [35S]streptavidin.
For all cell lines it was important to determine the window for optimal annexin V binding. Figure 5
illustrates the differences in time of induction of cell death, depending on the cell line and on the
inducer of apoptosis. In order to avoid the possibility of false negatives, the multiplexed assay with
crystal violet staining was useful to show if a decreased signal was due to significant loss of cells
capable of binding annexin V.
35
3500
7000
3000
2500
6000
2000
5000
4000
1500
0
8
35
8000
[ S]cpm for HUVE cells
[ S] cpm for H460 and
HCT 116 cells
4000
1000
16 24 32 40 48 56 64 72
Time (hours)
Figure 5. Measurement of apoptosis in (†) H460 cells („) HCT116 cells exposed to Apo-2L, and (z)
HUVE cells exposed to staurosporine(5), plotted as a time course after treatment.
An assay for the measurement of apoptosis has been developed using 96 well Cytostar-T scintillating
microplates. The assay simplifies the analysis of large numbers of samples, and is widely applicable to
a range of cell lines and treatment conditions. The homogenous assay format allows the immediate
measurement of early apoptotic events without further manipulation which may damage or remove
apoptotic cells.
References
1.
VERMEES, I et al., J. Immunol. Methods, 184, 39-51, (1995).
2.
WONG et al., J Cell Biochem., 60, 56-60, (1996).
3.
PITTI et al., J. Biol. Chem., 271, 12687-12690, (1996).
4.
KRAMER, S.M and CARVER, M.E., J. Immunol. Methods, 93, 201-206, (1986).
5.
McMURTREY et al., Cytotechnology, in press, 1999.
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Cytostar-T and Drop Design are trademarks of Amersham Biosciences Limited
Cytostar-T scintillating microplates are covered by US Patent No.5665562 and foreign equivalents
Amersham and Amersham Biosciences are trademarks of Amersham plc
Wallac and MicroBeta are trademarks of PerkinElmer, Inc
Scintillation Proximity Assay is covered by US Patent No 4568649, European Patent No 0154734 and
by Japanese Patent Application No 84/52452
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