Bodo Ortmann, Silke Valentin, Kristin Atze, Nadine Breuer, Steffi Franke, Mathias Kühn and Leon de Bruin
Lonza Cologne GmbH, Nattermannallee 1, 50829 Cologne, Germany

• Luminescence based calcium measurements in HUVECs
• Clonetics™ primary sensors utilized
• LUMIstar Omega microplate reader used to monitor dose-dependant calcium responses

Introduction
In drug discovery research cell-based assays are a widely accepted tool for high throughput screening of potential drug candidates. Typically, immortalized cell lines are used in cell based assays. They can be easily cultivated and modified in-vitro, but they bear some limitations as they are (1) often not originated from the actual tissue or native cell of interest, (2) sometimes non-human (e.g. hamster derived CHO cell line), and (3) often express the transfected targets at non-physiological levels.

For these reasons, there is a growing demand for many different primary human cells such as endothelial cells in drug screening and drug discovery research. Primary cells genuinely express the relevant drug targets, e.g. cell surface receptors at physiological levels, and carry the endogenous cofactors necessary for efficient and specific signal transduction. Primary cells provide model systems more closely resembling the in-vivo situation and allowing for a higher predictability of the situation in humans. In spite of this, they are rarely used for drug screenings. Main reasons may be the lack of assay formats suited for primary cells and the limited availability of reliable high quality primary cells.

Here, we have combined Lonza's expertise in providing reliable high quality primary cells with the Amaxa™ Nucleofector™ technology for highly efficient non-viral transfection of these cells without affecting their functionality to produce HUVEC calcium biosensor. This assay enables detection of intracellular Ca2+ fluxes upon external triggering of G-protein coupled receptors. We demonstrate that HUVEC calcium biosensor can be provided in a ready-to-use frozen state, and employed together with the LUMIstar Omega plate reader to generate pharmacologically relevant data on ligands of cell surface receptors.

Assay Principle

Fig. 1: Mechanism of the i-Photina® reaction

Incubation of cells, expressing the i-Photina® 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 and Methods

• White 96-well flat bottom plates from Corning (#3917)
• Ionomycin, ATP, thrombin, histamine were from Sigma Aldrich
• Neurotensin was from Bachem and mepyramine was from Tocris
• LUMIstar Omega, BMG LABTECH, Ortenberg, Germany
• Clonetics™ primary sensors - HUVEC calcium biosensor from Lonza

Production of HUVEC calcium biosensor
Clonetics™ human umbilical vein endothelial cells (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 using the LUMIstar Omega microplate reader
In order to perform the Ca2+ 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 from the top was carried out with the LUMIstar Omega microplate reader equipped with 2 automatic dispensers. Compounds (ionomycin, ATP, thrombin, histamine, and neurotensin) were injected into the wells. For antagonist experiments cells were preincubated for 5-15 min with mepyramine. Afterwards cells were stimulated with 7.5 µM histamine. Luminescence was recorded every second for 5 seconds prior to injection (base-line recording) and for a total of 30 seconds after injection of the compound.
Dose-dependent responses, EC50 and IC50 values were calculated using area under the curve (AUC) integration (GraphPad Prism).

Instrument settings LUMIstar Omega
Top Mode: luminescence – well mode (flash kinetic)
Temperature: 25°C
Injection: 25 µL (384-well), 50 µL (96-well)
Injection speed: 150 µL/s
Meas. time/well: 35 s
Interval time: 1.0 s
Integration time: 0.7 s
Gain: 3600
Emission: lens

Results and Discussion

Agonist stimulation
The LUMIstar Omega plate reader (Fig. 5) allows for following the kinetic reaction of the flash luminescence during the measurement (Fig. 2), injecting the stimulating agonist while recording of luminescence intensity is on-going. HUVEC calcium biosensor was reactivated from frozen state as described in Materials and Methods. Calcium release was triggered by injection of histamine (50 µM final concentration) in the LUMIstar Omega plate reader.

Fig. 2: Kinetics of HUVEC calcium biosensor flash luminescence upon stimulation with 50 µM histamine. The arrow indicates the time point of injection. The assay was performed in 96-well format as described in Materials and Methods.

Expression of functional photoprotein in HUVEC calcium biosensor was furthermore demonstrated by treatment with ionomycin. This pore-forming compound causes calcium flux in the cell in dose-dependent manner (Fig. 3).

Fig. 3: Dose-dependent responses of HUVEC calcium biosensor to a pore-forming compound and GPCR ligands. The assay was carried out in 96-well format as described in Materials and Methods. EC50: ionomycin 1 µM, histamine 6 µM, ATP 33 µM, neurotensin 8.8 nM

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 (Fig. 3).

Antagonist inhibition of the histamine response
The histamine response in HUVEC appears to be solely mediated through the H1 receptor as illustrated by complete abrogation of the histamine response through mepyramine, an inhibitor specific for the H1 receptor. Result is a clear dose-dependent response and an IC50 of 9.5 nM, in line with published data (Fig. 4).

Fig. 4: Histamine response of HUVEC is solely mediated through the H1 receptor. Mepyramine is an antagonist specific for the H1 receptor. The assay was performed in 384-well format as described in Materials and Methods.

Conclusion

Primary cells transiently transfected with the calcium biosensor i-Photina® are available as ready-to-use tool for drug discovery research. They are a groundbreaking robust (Z' = 0.6) new assay system for detecting intracellular Ca2+ dependent signalling upon stimulation with physiological agonists in high-throughput formats. This was demonstrated for four different classes of G-protein coupled receptors in dose-dependent manners for two plate formats. The LUMIstar Omega (Fig. 5) microplate reader is very well suited to reproducibly generate reliable data with low variability, to monitor dose-dependent Ca2+ responses and EC50/IC50 values consistent with published data.

For further information on HUVEC Calcium Biosensor see www.lonza.com/primarysensors

Fig. 5: LUMIstar Omega luminometer from BMG LABTECH

i-Photina® is a trademark of Axxam (Axxam, San Raffaele, Italy). Unless otherwise noted, all other trademarks herein are marks of the Lonza Group or its affiliates. The Amaxa™ Nucleofector™ technology is covered by patent and/or patent pending rights owned by Lonza Cologne GmbH. The i-Photina® luminescent calcium biosensor is covered by patent and patent pending rights owned by Axxam.