Study GPCRs Like a Pro

GPCRs (G protein-coupled receptors) are the largest

protein family linked to many normal biological as
well as pathological conditions. Also known as seven
transmembrane (7TM) receptors, the function of
GPCRs is highly diverse recognizing a wide range
of ligands, including photons, small molecules, and
proteins.
Molecular Devices offers a variety of assay and
instrument solutions to support studies of GPCR
function including assay kits, microplate readers,
washers, and handlers as well as cellular screening,
and imaging systems.

Study GPCRs Like a Pro

eBook Contents
Calcium flux assays ................................................................ 2
Ratiometric calcium assay ................................................... 3
Detection and quantitation of cAMP/cGMP .................. 4
GPCR targets in a high content screening
environment ............................................................................. 5
Photina luminescent calcium mobilization assays ...... 6
Cryopreserved Bacmam Transduced Aequorin Cells ... 7
Live cell Gi- and Gs-coupled GPCR second
messenger signaling ............................................................... 8
GPCRs systems and reagents .............................................. 9

For more information, visit:

www.MolecularDevices.com/GPCRs

Calcium flux assays

No wash protocol reduces well-to-well

variability, improving assay quality
(Z-factor) and reliability (CV %) of highthroughput screens

FLIPR Calcium Assay Kits apply a unique masking technology to reduce the background
fluorescence for detecting intracellular calcium changes in a simple and reliable
homogeneous assay format. They deliver pre-optimized, homogeneous, fluorescencebased formulations to expedite assay development and screening of GPCR and ion
channel targets.

Universal mix-and-read protocol accelerates

assay workflow and increases throughput

The kits are validated on the FLIPR Tetra High Throughput Cellular Screening System and
the FlexStation 3 Multi-Mode Microplate Reader.

Superior signal-to-noise ratio facilitates

confirmation of endogenous or transiently
transfected receptor activity during assay
development

Download Collateral
Calcium Assay Kits
Enhance Your Calcium Screens

Pre-optimized and validated protocols

ensure you can navigate both routine and
unconventional cell lines and targets

Calcium Signaling

FLIPR Calcium 6 Assay Kits provide the largest assay window

F/F
F
/F (max-min)
(max-min)

receptor

Calcium
6
Calcium

6
Calcium
6-QF
Calcium 6-QF
Calcium
5 5
Calcium
Fluo-4
Direct
Fluo-4
Direct
44M
MFluo-4W
Fluo-4W

4
3
2

Ca2+

Calcium 6

Ca2+

Significantly reduce
fluorescence background
with one-step protocol

Ca2+

-5

-4

-3

-2

-1

Log [carbachol] M

Calcium 6-QF
Quench-free option
for sensitive
targets and multiplexing

1
Increase in cytosolic Ca2+ can be detected by FLIPR or FlexStation

Comparison of FLIPR Calcium 6 Assay Kits to other calcium indicators

Ca 6
Ca6-QF
Ca 5
Fluo-4 Direct 4 uM Fluo-4W
was measured using agonism of the muscarinic receptor on CHO M1WT3
EC50 (nM)
16
17
23
20
21
cells from ATCC. Plates were read on either the FLIPR Tetra System or the
Z @ EC80
0.88
0.84
0.89
0.84
0.71
FlexStation
3 Microplate
Reader.
4
4.2
2.7
2.3
1.4
Signal window

2+
Increase in cytosolic
can
detecteddye
byindicators
FLIPR or FlexStation
MicroplateCa
Readers
usingbe
calcium-sensitive
Microplate Readers using calcium-sensitive dye indicators

Assay ready 1321N1 frozen cells

Anion exchange protein resistance

CHO-M1 cells in media: probenecid comparison

5

Calcium
Calcium 66

Calcium
PBXPBX
Calcium6 Kit
6 Kit

Calcium
6-QF
Calcium 6-QF

Calcium6 Kit
6 Kit
no PBX
Calcium
no PBX

Calcium
Calcium 55

F/F
(max-min)
F/F (max-min)

Baseline
(%)
Baseline (%)

Ca2+

Ca2+
sensitive
dye

Ca2+

L og [carbachol] M

Fluo-4 Direct
Fluo-4
Direct

300

200

100
0.001

Active Gq

Ca2+

400

Ca2+
sensitive dye

ligand

masking
dye

500

buffer

ligand binds to
cell-surface receptor

Calcium5 Kit
5 Kit
Calcium
PBXPBX
Calcium5 Kit
5 Kit
no PBX
Calcium
no PBX

0
0.01

0.1

10

LogLog
[histamine]
M
[histamine]
M

100

Assay ready
1321N1 frozen
cells, expressing
endogenous Histamine

Ca6
Ca6-QF
Ca5
Fluo-4 Direct
1 receptor
were
assayed
on
the
FlexStation
3
Microplate
Reader.
2.1
1.6
0.65
0.63
EC50 (M)
Comparison
of
histamine
receptor
response
to
increasing
concentrations
Signal window 5.2
4
3.2
3
of histamine demonstrates that the FLIPR Calcium 6 Assay Kit gives the
highest signal window.

www.MolecularDevices.com/GPCRs
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D:

-5

-4

-3

-2

-1

Log
M
Log[carbachol]
[carbachol] M

Novel fluorophore is more resistant to organic anion exchange protein,

Ca6+PBXenabling
Ca6 (no FLIPR
PBX)
Ca5+PBX 6 Ca5
(no PBX)
such as probenecid (PBX),
Calcium
Assay
Kit to produce
13
ND
50 (nM)
a stronger EC
signal
in the absence
of 39probenecid,14 while conserving
EC50
@ EC 80 at 0.9
0.92 pictured.
ND
values and ZZ-factors
EC80 > 0.5.0.86
CHO-M1 cells
2

R^2

Ratiometric calcium assay

The Fura-2 QBT Calcium Kit is a simple, mix-and-read format that employs our proprietary
masking technology with the industry-standard Fura-2 ratiometric calcium indicator to
accurately measure Gq-coupled GPCR mediated calcium mobilization. The kit eliminates the
cause of data variability and reduces the number of steps compared to conventional wash
protocols using Fura-2.
The kit is validated on the FLIPR Tetra High Throughput Cellular Screening System and the
FlexStation 3 Multi-Mode Microplate Reader.

See largest Fura-2 signal window available

Eliminate wash artifacts and increase
throughput with a homogeneous assay
Minimize the impact of uneven dye loading
and leakage on results
Interrogate low-density, weakly or nonadherent cells using a no-wash protocol

Download Collateral
Calcium Assay Kits
Homogenous Fura-2 Calcium Assay
Measuring Calcium Flux Assays

1.4

Antagonism
muscarinic
Antagonism of
of the
the muscarinic
M1M1
receptor
CHOM1
M1cells
cells
receptor on
on CHO
340/380 nm ratio (max.-min.)

340/380 nm ratio (max.-min.)

Agonism
of of
thethemuscarinic
M1
Agonism
muscarinic M1
receptor
ononCHO
receptor
CHOM1
M1 cells
cells
Fura-2 QBT

1.2

BD Kit
Fura-2 Wash

1.0
0.8
0.6
0.4
0.2
0.0

-5

-4

-3

-2

-1

1.4
1.2
Fura-2 QBT

1.0

BD Kit

0.8

Fura-2 Wash

0.6
0.4
0.2
0.0

-5

-4

Log [carbachol] M

-3

-2

-1

Log [atropine] M

The Fura-2
Calcium
the
largest
window and
most
atWash
EC80 or
CarbacholQBT Fura-2
QBT Kit provides
BD Kit
Fura-2
Wash signal Atropine
Fura-2
QBT robust
BDZ-factors
Kit
Fura-2
IC50 on
Reader.
EC50the
(nM)FlexStation
13 3 Microplate
17
19
2.4
2.2
3.3
IC50 (nM)
Z @ EC80

0.7

0.5

0.38

Z @ IC80

0.81

0.53

0.66

Window

1.1

0.85

0.7

Window

1.2

0.75

0.72

Agonism of endogenous H1
Agonism of endogenous H1 receptor
receptor on HeLa cells

Antagonism of endogenous H1

Antagonism of endogenous H1receptor

receptor on HeLa cells
on HeLa cells

Fura-2 QBT
BD Kit
Fura-2 Wash

0.8
0.6
0.4
0.2
0.0
-3

-2

-1

Log [Histamine] M

340/380 nm ratio (max.-min.)

340/380 nm ratio (max.-min.)

on HeLa cells

1.0

1.2
1.0

Fura-2 QBT
BD Kit
Fura-2 Wash

0.8
0.6
0.4
0.2
0.0
-4

-3

-2

-1

Log [pyrilamine] M

Histamine
QBTKitBD
Kit Fura-2
Wash
Pyrilamine
Fura-2
QBT signal
BD Kit
Fura-2robust
Wash
The Fura-2
QBTFura-2
Calcium
further
enhances
the assay by
providing
a larger
window,
EC
IC
(M)
0.2
0.09
0.3
(nM)
4.7
2.7
1.3
50 at EC80 or IC50, and removing assay variability by removing
50
Z-factors
wash steps. The assay was
Z @ EC
0.72
0.54Instrument
0.69 with UV LEDs.Z @ IC80
0.8
0.49
0.33
80
measured
using
the
FLIPR Tetra
Window

0.77

0.29

0.44

www.MolecularDevices.com/GPCRs

Window

1.1

0.52

0.37

Detection and quantitation of cAMP/cGMP

The CatchPoint cAMP and cGMP Fluorescent Assay Kits measure cAMP and cGMP levels,
and adenylate cyclase activity via a competitive immunoassay format. The kits high-affinity
reagents are optimized for sensitivity and precision in applications where cAMP and cGMP
levels are low. A single wash step removes unbound material prior to the development step,
so the assays are very resistant to interference from colored or fluorescent test compounds.
The kits are validated on the FlexStation 3, SpectraMax i3, SpectraMax Paradigm and
SpectraMax M Series Microplate Readers.

Download Collateral
CatchPoint cAMP Kit

CatchPoint cAMP and cGMP assay principle

HRP

Rapid Signal Developmentproprietary

Stoplight Red substrate generates a stable
and precise readout in only 10 minutes

HRP

HRP

No cAMP or cGMP
maximum HRP activity

Single wash step protocolresistant to

interference from colored or fluorescent
test compounds, improving reliability of
results

No Termination StepStoplight Red

substrate is fast and stable with a read
window out to 24 hours, providing flexible
read times without sacrificing data quality

CatchPoint cGMP Kit

HRP

Sensitive detection limit0.1 nM for cAMP

kit and 0.2 nM for cGMP kit

Increasing cAMP or cGMP

decreasing HRP activity

cAMP calibration curve

The calibration curve for the CatchPoint cAMP Assay Kit was generated
on the SpectraMax i3 Multi-Mode Microplate Reader. Data were taken
2 hours after addition of Stoplight Red substrate. The EC50 of the cAMP
calibration curve was 2.0 nM.

www.MolecularDevices.com/GPCRs

cGMP calibration curve

The calibration curve for the CatchPoint cGMP Assay Kit was generated
on the SpectraMax i3 Multi-Mode Microplate Reader. Data were taken
2 hours after addition of Stoplight Red substrate. The EC50 of the cGMP
calibration curve was 3.3 nM.

GPCR targets in a high content screening

environment

Works with all GPCRsknown and orphan

The Transfluor Assay is a cell-based fluorescence assay used to screen G-protein coupled
receptors (GPCRs), ligands, and other potential drugs that regulate GPCRs. By attaching
a fluorescent label to beta-arrestin, the location of the receptor-arrestin complex can be
monitored. Since desensitization only occurs with an activated receptor, monitoring betaarrestin translocation and subsequent receptor recycling provides a method to detect the
activation of any GPCR. This patented technology provides a powerful functional assay for
detecting a compounds activity against known and/or orphan GPCR targets. The Transfluor
Assay is optimized for HCS, secondary screening, and lead optimization.

Single read out for all GPCRs

No GPCR labeling or tagging
Requires no prior knowledge of interacting
G-protein
Eliminates the need for multiple GPCR
assays

The assay is validated on the ImageXpress Micro and ImageXpress Ultra High-Content
Analysis Systems.

Ideal for high content screening (HCS)

analysis

Download Collateral

Validated in over 100 GPCRs (from all

classes)

Transfluor Technology
Live Cell Kinetics Assay Utilizing the ImageXpress Micro System

Orphan GPCR Assay

Transfluor Assay principle

The Transfluor Assay utilizes the redistribution of beta-arrestin-GFP to

monitor GPCR activation and inactivation.

Agonist-independent assay used to verify the translocation

of arrestin-GFP in orphan GPCRs.

2AR-expressing cells

2AR-expressing cells were stimulated with isoproterenol. Left: control, center: pits, right: vesicles.
Transfluor assay imaged with the ImageXpress system.

www.MolecularDevices.com/GPCRs

Photina luminescent calcium mobilization assays

Calcium-activated photoproteins are important tools for detecting receptor-mediated
signaling events involving calcium mobilization in mammalian cells. One major advantage
of photoproteins is the immediate emission of flash luminescence upon calcium binding to
the coelenterazine-photoprotein complex. The background signal of Photina measurements
is close to zero, resulting in high signal-to-background ratios. Furthermore, the luminescent
light emitted by the oxidation of coelenterazine does not depend on optical excitation,
eliminating issues with auto-fluorescence.
This study provides a basic protocol for performing an adherent Photina assay using
the FlexStation 3 Multi-Mode Microplate Reader and FLIPR Tetra High Throughput
Cellular Screening System with ICCD camera. Both instruments were used to determine
the concentration response of IMETIT in CHO mito-Photina/H3 cells at various cell
concentrations.

Agonist response measured with FLIPR Tetra System with ICCD camera option
CHO-H3 Photina Cell Titration

Available with an aequorin option

including a novel ICCD camera technology
optimized for use with both fluorescent and
luminescent assays
Cell suspension system makes it amenable
to both adherent and suspension cell-based
assays in 96-, 384- and 1536-well formats

Permits real-time measurement of

fluorescent & luminescent cell-based assays
in 96- or 384-well formats
Up to one column of the plate can be
monitored simultaneously before, during,
and after compound addition

12000

RLU (Max. - Min.)

Flexible HTS/uHTS solution for early

identification of lead compounds in the
drug discovery process

FlexStation 3 Reader

Download Application Note

14000

FLIPR Tetra System

10000
8000

Disposable pipette tips reduces crosscontamination, saving precious reagents

6000
4000
2000
0
1e-5

1e-4

0.001

0.01

0.1

IMETIT Concentration (uM)

CHO mito-Photina/H3 cell assay. (5000 ( ), 2500 ( ), 1250 ( ), and 625 ( ) cells/well). FLIPR Tetra
System with ICCD camera option added agonist during luminescent read mode. Results are the
average of approximately 32 replicates.

Agonist response measured with FlexStation 3 System

CHO-H3 Photina Cell Titration

12000

RLU (Max.-Min.)

10000
8000
6000
4000
2000
0
1e-5

1e-4

0.001

0.01

0.1

IMETIT Concentration (M)

CHO mito-Photina/H3 cells were plated at varying cell concentrations (5000 ( ), 2500 ( ), 1250
( ), and 625 ( ) cells/well) in 384-well, black-wall, clear-bottom plates. The FlexStation 3 System
added agonist during real-time luminescent detection. Results are the average of approximately
16 replicates.

www.MolecularDevices.com/GPCRs

Cryopreserved Bacmam Transduced

Aequorin Cells
Aequorin is a photo-sensitive protein that emits luminescent light in response to calcium.
Cryopreserved cells as reagents in Aequorin based calcium flux assays decouples the tissue
culture process from high throughput screening while improving overall assay performance.
The need for culturing cells in plates is eliminated.
This study demonstrates performance of cryopreserved Bacmam transduced Aequorin cells in
384- well and 1536-well formats. Combined with the FLIPR Tetra High Throughput Cellular
Screening System equipped with Aequorin options, cryopreserved cells are a powerful tool in
the identification of lead compounds in drug discovery.

Download Poster

Aequorin-based suspension assays with

frozen cells demonstrate both instrument
and cell performance during extended
assays without significant shift in EC50 or
Z factor
The EC50 values remain within one half
log of expected results and there is little
reduction in signal intensity over time

Z factor over time during Bacmam Aequorin

Suspension Cell Assays

Z Factor

RLU (max-min)

Cell Titration and CRC Assay measured with

FLIPR Tetra System

Cryopreserved Bacmam transduced Aequorin

cell lines can be used to produce robust
assay results in both 384-well and 1536well formats on the FLIPR Tetra System with
Aequorin options

[Agonist] nM

In a 384-well plate, frozen Bacmam Transduced Aequorin Cells were

titrated against a CRC of target agonist. In this assay, cells were preplated in suspension prior to addition of compound.

Time (min)

Recording of whole plate Z factor during extended plate screening

assays. 384-well plates contained 22 columns of EC80 reference
compound and 2 columns of negative controls. Average Z factor = 0.7.

RLU (AUC)

Change in signal over time during Bacmam Aequorin

Suspension Cell Assays

Time (min)

Change in RLU over time during six hour 384-well suspension cell
experiment. Cells were added at 2,500/well.

www.MolecularDevices.com/GPCRs

Live cell Gi- and Gs-coupled GPCR second

messenger signaling
Detection of Gi- and Gs-coupled GPCR second messenger signal activity has been
traditionally accomplished using assays such as radioactive binding or endpoint cAMP assays
that require cell lysis. Such assays measure activity at a single time point in the cellular
response and do not provide kinetic information. Another option utilizes forced-coupling
of Gi- and Gs-GPCRs to G16 followed by fluorescence detection of calcium flux upon
agonist receptor activation. Again, this assay is sub-optimal as it does not signal through the
biorelevant cAMP pathway.

GloSensor cAMP Assay is demonstrated

on the FLIPR Tetra System as a live cell
HTS screening application for Gi- and Gscoupled GPCRs
U
 se of the FLIPR Tetra System with
GloSensor cAMP Assay enables kinetic
measurement of Gi- and Gs-coupled
receptor signaling not possible using
endpoint assays on standard plate readers
A
 ssay development flexibility using
GloSensor cAMP cell lines and plasmids
transfected in endogenous as well as stably
transfected receptor cell lines

In this study we demonstrate endogenous receptor activity in CHO-K1 and HEK-293 cell
lines stably expressing the GloSensor plasmid using the FLIPR Tetra High Throughput
Cellular Screening System.

Download Application Protocol

HEK-293 cells over expressing Gi-coupled D4 receptor

2000

25000

1500

20000

RLU (max-min)

RLU (Minimum)

Gi-coupled agonsim

1000
500
0

15000
10000
5000
0
-12
-11 -10 -9 -8 -7 -6

-4 -2 0 2

Log [Dopamine] M

Log [Peptide YY(3-36)] M

Gi-coupled GPCR receptor agonism results in a reduction in signal

correlated with reduction in cAMP inside the cell. Baseline increase in
cAMP activity was induced by the addition of forskolin. Using stable P2Y
receptor in CHO-K1 cells, we compared results upon addition of forskolin
either before or after the agonist. 10 M forskolin addition followed 15
minutes later by addition of agonist Peptide YY(3-36) on the FLIPR Tetra
System.

HEK-293 cells over expressing Gi-coupled dopamine D4 receptor from

Multispan, Inc. were transiently transfectd with GloSensor cAMP-22F
plasmid. Ligand was added on-line to the wells, followed by 5 minute
incubation. Continuing the assay, FLIPR Tetra System added 10 M
forskolin to stimulate cAMP production in the cell. Inhibition of forskolin
mediated cAMP production by Dopamine.

Gs-coupled GPCR agonist and antagonist

A

B
30000

30000

RLU (max-min)

RLU (max-min)

40000

20000
10000
0
-11
-10 -9 -8 -7 -6 -5

Log [Isoproterenol] M

20000

10000

0
-12
-11 -10 -9 -8 -7 -6 -5

Log [Propranolol] M

Transient transfection of GloSensor cAMP-22F and endogenous Gs-coupled cAMP response in HEK
293 cells. (A) Response to isoproterenol and (B) inhibition of the response to 100 nM isoproterenol
by propranolol. Results are comparable to those obtained from the experiment performed with the
stable GloSensor HEK-22F cell line.

www.MolecularDevices.com/GPCRs

GPCRs Systems and Reagents

For detailed information, select the images or text.

Reagents
FLIPR Calcium Assay Kits
Fura-2 QBT Calcium Kit
CatchPoint cAMP
Fluorescent Assay Kit
FLIPR Tetra High Throughput
Cellular Screening System

CatchPoint cGMP
Fluorescent Assay Kit

FlexStation 3 Multi-Mode
Microplate Reader

Reagents
CatchPoint cAMP
Fluorescent Assay Kit
CatchPoint cGMP
Fluorescent Assay Kit
SpectraMax i3 Multi-Mode
Microplate Reader

SpectraMax Paradigm
Multi-Mode Microplate Reader

SpectraMax M Series
Multi-Mode Microplate Reader

Reagent
Transfluor Assay

ImageXpress Micro XLS

Widefield High-Content
Analysis System

ImageXpress Ultra Confocal

High-Content Analysis System

www.MolecularDevices.com/GPCRs
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2014 Molecular Devices, LLC | 10/14 Version 1 | Patents: www.moleculardevices.com/patents