Download Monitoring Intracellular cAMP with hMSC cAMP

Monitoring Intracellular cAMP with hMSC
cAMP biosensor using the LUMIstar Omega
K. Atze1, B. Ortmann1, S. Valentin1, M. Kühn1, S. Franke1, N. Breuer1, P. Stecha2, B. Binkowski2, L. de Bruin1, A. Pitt1
Lonza Cologne GmbH, Nattermannallee 1, 50829 Cologne, Germany 2Promega Corporation, Madison, WI 53711 USA
1
Application Note 221, Rev. 01/2012
Assay Principle
Luminescence based cAMP measurements in hMSC
Poietics™ Primary Sensors utilized
LUMIstar Omega microplate reader used to monitor dosedependent cAMP responses
Firefly luciferase has been fused genetically to the cAMP-binding
domain of human Protein kinase A (red). Upon binding of cAMP
the whole protein molecule undergoes a conformational change.
This activates the luciferase domain which converts luciferin to
oxyluciferin and emits luminescence. For more information see
www.promega.com/glosensor.
Introduction
Primary cells allow for a higher predictability of drug reactions in
humans. These cells endogenously express relevant drug targets
at physiological level and genuinely carry the components required
for specific signal transduction. They can be derived from the actual
tissue of interest. These are significant advantages over immortalized
cell lines, which may be derived from irrelevant tissue, be of nonhuman origin, and often express transfected drug targets at nonphysiological levels. For these reasons, there is a growing demand for
primary cells in drug screening and hit validation.
Here we show that primary human mesenchymal stem cells can be
used in high-throughput formats (i.e. 96-well and 384-well plate)
to monitor changes of intracellular cAMP concentration. The cells
transiently express Promega’s luminescent live-cell biosensor
GloSensor™ and can be cryopreserved without loss of functionality.
After reactivation of the cells and loading with the GloSensor™
substrate the assay is ready to use. The functional expression
of the GloSensor™ as well as the functionality and specificity of
endogenously expressed G-protein coupled receptors triggering
intracellular cAMP production was shown through receptor-binding
agonists in dose-dependent manner. A Z’ value of 0.7 facilitates the
use in high-throughput screenings. We also provide evidence for a
versatile assay system, cells co-expressing the calcium biosensor
i-Photina® and the cAMP biosensor. Both signaling pathways can
consecutively be monitored in one sample.
The LUMIstar Omega microplate luminescence reader is very well
suited to monitor dose-dependent cAMP responses, to generate EC50
values consistent with published data and to reproducibly produce
reliable data with low variability.
Fig. 2: Mechanism of the GloSensor™ reaction
Materials and Methods
96-well and 384-well white flat bottom plates from Corning
pGloSensor™22F cAMP plasmid & GloSensor cAMP reagent from
Promega
LUMIstar Omega, BMG LABTECH, Ortenberg, Germany
Poietics™ human mesenchymal stem cells (hMSC) from Lonza
Production of hMSC cAMP biosensor
Poietics™ human mesenchymal stem cells (hMSC) were transiently
transfected with an expression plasmid encoding the GloSensor™22F using the Amaxa™ 96-well Shuttle™ Nucleofector™.
The transfected hMSCs were incubated after Nucleofection™ in a
humidified tissue culture incubator (37°C, 5 % CO2) for 6 hours.
Subsequently the cells were frozen in vials in cryoprotective agent.
Detection of intracellular cAMP using the LUMIstar Omega
microplate reader
In order to perform the cAMP-assay cryopreserved cells were thawed,
seeded on a 96-well or 384-well plate, and were allowed to recover
over night. 4 hours after thawing medium was exchanged for HEPESbuffered medium to remove the cryoprotective agent.
Measurement of luminescence was carried out with the LUMIstar
Omega microplate reader equipped with 2 automatic dispensers.
Compounds (forskolin, isoproterenol, histamine from Sigma
Aldrich; prostaglandin E1 from Calbiochem) were added to the
wells. Luminescence was recorded every 5 minutes for 5 seconds.
Luminescence was recorded for a total of 25 minutes. Dose-dependent
responses and EC50 values were calculated by GraphPad Prism®
using the peak values.
Fig. 1: BMG LABTECH’s LUMIstar Omega multidetection microplate reader
Results and Discussion
Normalized Luminescence [%]
Assay performance
The functional expression of the GloSensor™ protein in hMSC
reactivated from frozen state was demonstrated by treating
the cells with forskolin, a substance that stimulates the
adenylyl cyclase to convert ATP to cAMP. Result is a clear dosedependent response with an EC50 consistent with published data
(Fig. 3). Furthermore, by applying ligands of G-protein coupled
receptors genuinely expressed on the cell surface the cAMP pathway
is triggered demonstrating proper functioning of this signaling
pathway in hMSC after freezing and reactivation.
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A
50 µM Histamine + 5 µM Isoproterenol
50 µM Histamine
5 µM Iroproterenol
Water control
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Time (s)
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50 µM Histamine + 5 µM Isoproterenol
50 µM Histamine
5 µM Iroproterenol
Water control
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Fig. 5: cAMP biosensor and Ca2+ biosensor can be multiplexed to monitor 2
pathways in one sample.
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Formoterol, EC50: 0.9 nM
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log [agonists] (M)
Forskolin, EC50: 3.2 µM
PGE1, EC50: 177 nM
Fig. 3: Dose-dependent responses of hMSC cAMP biosensor to different
stimulants.
Demonstrating the uniformity of the luminescence signal across
several samples cells were treated in alternating columns with either
forskolin or medium as control (Fig. 4). Results were a low coefficient
of variability (CV) of 10% for the forskolin stimulated samples, and a
high fold induction of 21 yielding a sizable assay window. The overall
robustness of this assay format in conjunction with the LUMIstar
Omega is demonstrated by a Z’ value of 0.7.
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peak (RLU)
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Luminescence (RLU)
For production of Clonetics™ Primary Sensors expressing the Ca2+
biosensor i-Photina® and performing Ca2+-dependent assays see
BMG LABTECH application note No. 216 (http://www.bmglabtech.
com/application-notes/luminescence/luminescence-calciumbiosensor-216.cfm).
Multiplexing the assay
Cells expressing both Ca2+ and cAMP biosensors were stimulated
with either histamine (triggering Ca2+ signaling) or isoproterenol or
with a combination of both agonists. The fact that i-Photina®
generates flash luminescence and GloSensor™ a more slowly
increasing glow luminescence allows the monitoring of both
signaling pathways in one sample. This is shown in Fig. 5 A (recorded
i-Photina® flash luminescence) and B (detected GloSensor™ glow
luminescence) where histamine and isoproterenol applied alone
generate luminescence in the respective time window (in A
or B, resp.), the samples treated with the combination of both emit
flash and glow luminescence consecutively.
Luminescence (RLU)
Instrument settings
Mode: luminescence – end point measurement
Temperature: 25 °C
Interval time: 5 min
Integration time: 5 sec
Number of cycles: 5 or 6
Gain: 2000 or 3600
30000
Human mesenchymal stem cells transiently transfected with the
live-cell cAMP biosensor GloSensor™ are available as ready-to-use
tool for drug discovery research. They are a groundbreaking robust
new assay system for detecting intracellular cAMP-dependent
signaling upon stimulation with physiological agonists in highthroughput formats leaving the cells intact and alive. Multiplexing
cAMP biosensor and Ca2+ biosensor enables the detection of
both signaling pathways in one sample saving time and costs.
The ready-to-use cell based assay system in conjunction with the
LUMIstar Omega plate reader is an excellent tool to evaluate drug
effects on signaling in primary cells.
The live-cell biosensor GloSensor™ is a trademark of Promega (Promega Corporation,
Madison, WI, USA) and is covered by patent and patent pending rights owned by Promega.
i-Photina® is a trademark of Axxam (Axxam SpA, Milan, Italy) and is covered by patent and
patent pending rights owned by Axxam. The Nucleofector™ Technology is a trademark
of Lonza (Lonza Cologne GmbH, Koeln, Germany) and is covered by patent and/or patent
pending rights owned by Lonza.
For further information on primary sensors and other related products, please visit www.
lonza.com/drugdiscovery or www.lonza.com/primarysensors.
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96-well columns
Fig. 4: 3 hMSC cAMP biosensor yields luminescence intensities across a
microplate with only small variations and a Z’ value of 0.7.
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