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 non physiological levels. For these reasons, there is a growing demand for primary cells in drug screening and hit validation.
In this application note 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. For the monitoring of Ca2+ fluxes human umbilical vein endothelial cells (HUVEC) proved to be useful.
Assay Principle
cAMP Biosensor assay
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.
Calcium Biosensor assay
Incubation of cells, expressing the i-PhotinaR apo-photoprotein, with coelenterazine in the presence of oxygen leads to formation of a stable complex, the active photoprotein. Calcium released from intracellular stores upon stimulation of the cells with agonists via G-protein coupled receptors binds to the photoprotein. The excited photoprotein converts coelenterazine to coelenteramide and emits a flash of blue luminescence.
Materials & Methods
- 96-well and 384-well white microplates from Corning
- pGloSensor™22F cAMP plasmid & GloSensor cAMP reagent from Promega
- Poietics™ human mesenchymal stem cells (hMSC) from Lonza
- Ionomycin, ATP, thrombin, histamine from Sigma Neurotensin was from Bachem
- CloneticsTM primary sensors - HUVEC calcium biosensor 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.
Production of HUVEC calcium biosensor
Clonetics™ HUVEC were transiently transfected with an expression plasmid encoding i-Photina® using the appropriate Amaxa™ 96-well Nucleofector™ kit and the Amaxa™ 96-well Shuttle™ Nucleofector. The transfected HUVECs were incubated after Nucleofection™ in a humidified tissue culture incubator (37°C, 5 % CO2) for 6 hours. Right before freezing the cells were loaded with 10 µM native coelenterazine for 2 hours. Subsequently the cells were frozen in vials in cryoprotective agent.
Detection of intracellular calcium release or intra-cellular cAMP
In order to perform the Ca2+-assay or 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. The plate was then ready to be measured.
| Calcium assay | |||
| FLUOstar/ LUMIstar Omega | CLARIOstar | PHERAstar FS | |
| Detection mode | Luminescence, plate mode |
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| Method | Endpoint, Top optic |
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| Optic settings | Lens | monochromator full range | luminescence specific Optic module |
| Cycle time | 5 min |
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| No of cycles | 5-6 | ||
| Integration time | 5 sec |
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| Temperature | 25°C |
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| cAMP assay | |||
| FLUOstar/ LUMIstar Omega | CLARIOstar | PHERAstar FS | |
| Detection mode | Luminescence, well mode |
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| Method | Endpoint, Top optic |
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| Optic settings | Lens | monochromator full range | luminescence specific Optic module |
| Temperature | 25°C | ||
| Injection | 25 µl (384-well), 50 µl (96-well) | ||
| Injection speed | 150 µl/sec | ||
| Meas. time/well | 35 sec | ||
| Interval time | 1 sec | ||
| Integration time | 0.7 sec | ||
Results & Discussion
Ca2+ Assay
The reaction of the flash luminescence was followed over time (Fig. 3). Injecting the stimulating agonist while recording of luminescence intensity is on-going. Calcium release was triggered by injection of histamine (50 µM final concentration).
The assay system is well suited to generate pharmacologically relevant data for compounds that trigger calcium dependent signalling. Agonists for different classes of GPCRs (histamine receptors, purinergic receptors, neurotensin receptors) clearly show dose-dependent responses with the HUVEC calcium biosensor yielding EC50 values consistent with published data (data not shown).
cAMP Assay
The functional expression of the GloSensor™ protein in hMSC reactivated from frozen state was demonstrated by treating the cells with forskolin. Result is a clear dose-dependent response with an EC50 consistent with published data (Fig. 4).
A linear relationship between protein concentration and fluorescence units is obtained indicating that small concentrations of protein in samples can be determined using the BMG LABTECH microplate reader.
Conclusion
The ready-to-use cell based assay systems from Lonza in conjunction with a microplate reader from BMG LABTECH are an excellent tool to evaluate drug effects on signaling in primary cells.