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
1Lonza Cologne GmbH, Nattermannallee 1, 50829 Cologne, Germany 2Promega Corporation, Madison, WI 53711 USA

• Luminescence based cAMP measurements in hMSC
• Poietics™ Primary Sensors utilized
• LUMIstar Omega microplate reader used to monitor dose-dependent cAMP responses

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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 non-human origin, and often express transfected drug targets at non-physiological 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. 1: BMG LABTECH's LUMIstar Omega multidetection microplate reader

Assay Principle
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.

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 HEPES-buffered 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.

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

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-calcium-biosensor-216.cfm).

Results and Discussion
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 dose-dependent 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.

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.

Fig. 4: 3 hMSC cAMP biosensor yields luminescence intensities across
a microplate with only small variations and a Z' value of 0.7.

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.

Fig. 5: cAMP biosensor and Ca2+ biosensor can be multiplexed to monitor 2 pathways in one sample.

Conclusion
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 high-throughput 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.