Running an Aequorin Cell-Based Luminescent Assay on the FLIPR TETRA

Running an Aequorin Cell-Based Luminescent Assay on the FLIPRTETRA®
Veena Menon1, Suresh Babu Poda1, Natalia Betancourt1, Kathy Zhao2 and Vincent Dupriez2
1 Lundbeck Biological Research USA, Inc and 2 PerkinElmer Life & Analytical Sciences
Kinetic measurement of Aequorin signal on
the FLIPRTETRA®
4
Kinetics of CHO-Aequorin Response - FLIPRTETRA®
Reagents
Pe
eak RLU
10 000 cells/well, black, clear bottom assay plate
500
Ligand
Dispensing
3000
Double transfected aequorin cell lines, stably expressing both mitochondriatargeted aequorin and a GPCR were used in this study: Histamine H1
AequoScreen® cell line (PerkinElmer # ES-390-A), Vasopressin V1B
AequoScreen®; cell line (PerkinElmer # ES-362-A), Vasoactive Intestinal Peptide
VPAC1 AequoScreen® cell line (PerkinElmer # ES-273-A). In addition, the CHOK1 (+Gα16) AequoScreen® Parental cell line (PerkinElmer # ES-000-A24 ) was
also used
used, using ATP as an agonist of the endogenous P2Y receptor.
receptor
Assay Buffer was DMEM/Ham's F12 culture medium (with 15 mM HEPES, Lglutamine, without phenol red; Invitrogen Cat n°11039) + 0.1% protease-free
BSA. Coelenterazine h came from Promega (Cat n° S2011): 1 mM stock solution
in methanol. Digitonin came from Sigma (Cat n°37006): 50 mM stock solution in
DMSO
DMSO.
Kinetics of CHO-Aequorin-H1 Response - FLIPRTETRA®
2000
1000
0
0
5
10
15
ATP (-3)
ATP (-4)
400
ATP (-5)
ATP (-6)
300
ATP (-6.5)
200
ATP (-7)
( 7)
ATP (-7.5)
100
ATP (-8)
Buffer
0
Digitonin 100 µM 0
Pe
eak RLU
2
10 000 cells/well, black, clear bottom assay plate
4000
20
25
time (s)
30
35
40
45
Ligand
Dispensing
5
10
15
20
25
30
35
40
45
Histamine (-6)
Histamine (-7)
Histamine (-8)
Histamine (-8.5)
Histamine ((-9)
9)
Histamine (-9.5)
Histamine (-10)
Buffer
Digitonin 100 µM
time (s)
5 000
0
-10
-9
-8
-7
-6
-5
-4
-3
Z' (EC100 Ago vs Buffer)
6.49
6.4
6.42
6.45
0.85
0.94
0.47
0.61
3 000
1 500 000.0
Histamine
Digitonin
ATP 100 µM
Buffer
2 000
1 000
pEC50 = 9.19
Z' (EC80) = 0.62
AUC (0
0-60sec)
10 000 c/w
5 000 c/w
2 000 c/w
1 000 c/w
pEC50
AUC (Samp
ples 5 to 14)
10 000
LumiLux®
5 000 Adherent Frozen Cells/Well
FLIPRTETRA®
10 000 Adherent Frozen Cells/well
10 000 c/w
5 000 c/w
2 000 c/w
1 000 c/w
15 000
Pharmacology (FLIPRTETRA® vs LumiLux®)
H1
CHO (+Gα16) Aequorin Parental Cell Line
1 000 000.0
pEC50 = 9.18
Z' (EC80) = 0.61
500 000.0
0
0.0
-13 -12 -11 -10 -9
-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2
-2
Histamine
Digitonin
ATP 100 µM
Buffer
-8
-7
-6
-5
-4
-3
Log[Ligand],M
Log[ligand],M
Log[ATP],M
CHO-Aequorin-H1 Cell Line
10 000 c/w
10
000 c/w
5 000 c/w
2 000 c/w
1 000 c/w
1 000
500
0
-11
-500
-10
-9
-8
-7
-6
pEC50
Z' (EC100 Ago vs Buffer)
9.42
9.27
9.28
9.05
0.66
0.85
0.5
< 0
V1B
Ph
Pharmacology
l
(FLIPRTETRA® vs LumiLux
L iL ®)
FLIPRTETRA®
10 000 Adherent Frozen Cells/Well
4 000
LumiLux®
5 000 Suspension Frozen Cells/Well
Arg-8-Vasopressin
Digitonin
ATP 100 µM
Buffer
3 000
2 000
pEC50 = 10.40
1 000
Z' (EC80) = 0.69
Log[ligand],M
LumiLux®
5 000 Suspension Frozen Cells/Well
FLIPRTETRA®
10 000 Adherent Frozen Cells/Well
-5
Log[Histamine],M
Increasing cell numbers per well were used to assess the sensitivity of
the FLIPRTETRA® in relation with the brightness of the cell lines. EC50
values were unchanged by varying the cell density. The CHO (+Gα16)
Aequorin parental cell line was brighter than the H1 cell line, and
yielded acceptable values even when using 1,000 cells/well. For the H1
cell line, at least 2,000 cells/well were needed to get sufficient signal
intensity.
6A
VPAC1
2 000 000
Arg-8-vasopressin
ATP 100 µM
Buffer
1 500 000
pEC50 = 10.3
1 000 000
Z' (EC80) = 0.60
500 000
0
-15
15 -14
14 -13
13 -12
12 -11
11 -10
10 -9
9 -8
8 -7
7 -6
6 -5
5 -4
4
Log[ligand],M
CHO-Aequorin-Vasopressin 1B receptor cells were analyzed on the
FLIPRTETRA® and on the LumiLux® Cellular Screening Platform using
the adherent (FLIPR®) and suspension (LumiLux®) luminescence assay
protocols EC50 values obtained on both readers and in both assay
protocols.
modes were equivalent.
VIP
Digitonin
ATP 100 µM
Buffer
2 500
2 000
AUC
(0-60sec)
1 500
10 000 c/w
5 000 c/w
2 000 c/w
1 000 c/w
CHO-Aequorin Histamine H1 receptor AequoScreen® cells were analyzed
on the FLIPRTETRA® and on the LumiLux® Cellular Screening Platform
using the adherent luminescence assay protocols. EC50 values obtained
on both readers and in both assay modes were equivalent.
AUC
(samples 5 to 14)
2 000
0
-15-14-13-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 -1
Using 3 second intervals, kinetics of the emission of light can be
measured by the FLIPRTETRA®. The kinetics of the responses of 2
AequoScreen® cell lines are shown. The signal intensity was well
above 150 Peak RLU (dashed line), which is an acceptable
th h ld lilimit.
threshold
it
6B
Number of cells per well - sensitivity
AUC (0-60sec)
compounds resulting in a large percentage of false positive or false
negative hits in agonist and antagonist screens respectively.
D di t d hi
Dedicated,
highly
hl sensitive
iti readers
d
such
h as th
the L
LumiLux
iL ®
®
(PerkinElmer), the MicroBetaJet (PerkinElmer), the FDSS
(Hamamatsu Photonics) and the Cybi®Lumax (Cybio) have already
been in use for several years to perform drug discovery research
g aequorin
q
assays.
y Here,, we show that selected aequorin
q
cell
using
lines generating intense luminescent signal allow comfortable
detection with a less sensitive, non-luminescence dedicated reader,
i.e. the FLIPRTETRA® (Molecular Devices). The FLIPRTETRA® was built
for fluorescent signal detection, but by putting off the excitation light,
can also be used for strong luminescent signal detection
detection. We show
several examples of aequorin signal detection with the FLIPRTETRA®,
generating high quality data, with Z’ values compatible for HTS use
of Aequoscreen® assay on the FLIPRTETRA®.
Adherent cells assay: For FLIPRTETRA® measurements, cells were tested mainly in adherent
mode (10 000 cells/well unless otherwise indicated), but some suspension cell assays were
also performed. Cells (frozen vial) were thawed and plated in TC-treated assay plates (in
Ham’sF12 with 10% serum, no antibiotics) and left in the incubator overnight (37°C, 5%
CO2). The next day, medium was removed by plate overthrow and tapping on a paper towel,
then 20 μL/well of Assay Buffer containing 10 μM coelenterazine h was added to the cells
and plates were incubated for 4h at RT° in the dark. Ligands (20μL/well), diluted in Assay
B ff were dispensed
Buffer,
di
d on the
h cells
ll using
i the
h FLIPR®. We
W usedd black,
bl k clear
l bottom
b
384384
well assay plates as we observed a background signal that was variable from well to well
when using white assay plates (not shown).
Suspension cells assay: For LumiLux® measurements, cells were tested in suspension mode
(5 000 cells/well). Cells (frozen vial) were thawed and resuspended in 10-ml of assay buffer
containing
co
ta
g 5 μM
μ coe
coelenterazine
e te a e h.. Thiss ce
cell suspension
suspe s o was put in a 10-ml
0
Falcon
a co tube,
fixed onto a rotating wheel and incubated for 4 h or overnight at RT° in the dark (8 rpm; 45°
angle). Cells were diluted with Assay Buffer to 5 000 cells/20 µL. Ligands (20μL/well),
diluted in Assay Buffer, were prepared in black, clear bottom assay plates, and the cell
suspension was dispensed on the ligands using the LumiLux®.
Digitonin at a final concentration of 100 µM diluted in Assay Buffer was used to measure
th receptor-independent
the
t i d
d t cellular
ll l calcium
l i
response (cell
( ll membrane
b
permeabilization).
bili ti )
The “Top/digitonin” response (expressed in percentage) is the ratio between the maximal
response to the ligand of the receptor (Top) and the digitonin response (Digitonin), which is
indicative of the aequorin content of the cells.
FLIPRTETRA® settings: For using the FLIPRTETRA® in luminescence mode, the excitation
light
g was disabled ((select “Exc.WLength:
g NONE” in the software),
), a ggain of 240 and an
exposure time of 3 seconds were used. 20 µL of ligands were dispensed form the source
plate 1 to the read plate containing the coelenterazine-loaded cells. 33 intervals of 3.1
seconds were read, 3 of them being read before ligand dispensing.
Area under the curve (AUC) was calculated with the ScreenWorks™ software, using the
third interval for background subtraction (“subtract bias based on sample 3”).
AUC (Sa
amples 4-33)
Aequorin based Ca2+ assays
Aequorin-based
represent a new paradigm in drug
discovery research for Ca2+-coupled
GPCRs and ion channels cell-based
assays. The inherent limitations of
fl
fluorescent
t dye-based
d b
d screening
i
result in lower throughput and higher
resource expenditure in comparison
to the aequorin assay. Furthermore,
g the fluorescence
in a HTS setting
assay approach is prone to
interference from autofluorescent
5
AequoScreen® assay
AUC (Sample
es 5-14)
3
Introduction
AUC (samples 5 to 1
14)
1
1 500
pEC50 = 8.67
1 000
Z' (EC80) = 0.58
500
400 000
VIP
Digitonin
ATP 100 µM
Buffer
300 000
200 000
pEC50 = 7.90
100 000
Z' (EC80) = 0.71
0
-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2
0
-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
Log[ligand],M
Log[Ligand],M
CHO-Aequorin Vasoactive Intestinal Peptide VPAC1 receptor
AequoScreen® cells were analyzed on the FLIPRTETRA® and on the
L iL ® Cellular
LumiLux
C ll l S
Screening
i Pl
Platform
tf
using
i th
the adherent
dh
t (FLIPR®) and
d
®
suspension (LumiLux ) luminescence assay protocols. EC50 values
obtained on both readers and in both assay modes were equivalent.
7
Conclusion
We have shown here that signal intensity for the selected catalog
aequorin cell lines is strong enough to allow its measurement by the
non-luminescence dedicated FLIPRTETRA®. In particular,
particular signal intensity
of the CHO (+Gα16) aequorin parental cell line was very high, and thus
will undoubtedly generate double-transfectant cell lines that will be
suitable to use on the FLIPRTETRA®. Pharmacology and Z’ values
observed are fully compatible with the use of these cell lines for HTS or
profiling
fili
studies.
di
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