4 DEVELOPMENT OF FUNCTIONAL ASSAYS FOR THE HUMAN NPY Y 2
4.3 Other techniques to measure a calcium response in CHO-hY 2 -K9-qi5-K9-mtAEQ-A7 cells
4.3.1 Introduction
Confocal microscopy has often been used for the measurement of intracellular calcium concentrations in single cells (Lipp et al., 2001). However, the highly sophisticated instrumentation makes this technique not very well suited for the application in a functional assay for routine compound screening. In addition, single cell measurements can vary to a high degree depending on the sample preparation which is unfavorable for quantitative determinations. Nevertheless, confocal microscopy is a useful tool for the visualization of changes in intracellular calcium.
The use of plate readers equipped with a CCD camera for the detection of flash luminescence has already been described (Dupriez et al., 2002). Therefore, it was investigated whether the light generated by the transfected CHO cells upon agonist binding was sufficient for the detection with a CCD camera.
4.3.2 Materials and methods
4.3.2.1 Confocal microscopy
CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells were seeded in 200 µl Ham’s F12 + 10 % FCS on a Lab-Tek® II, 8 chamber coverglass system (Nalge Nunc, Naperville, IL, USA) two days prior to the experiment and were grown to 50-70 % confluence. 330 µl of
loading suspension were prepared as described in section 4.1.2.4 and added to 1 ml of L-15 Leibovitz medium (Sigma). The medium of the cells was displaced with 300 µl of this solution and the cells were incubated for 30 min at room temperature. Then, cells were washed once with loading buffer (see section 4.1.2.4) and incubated for 30 min with 270 µl of Leibowitz medium at room temperature. The chamber was installed into a Zeiss Axiovert 200 M microscope, equipped with the LSM 510 laser scanner using a Plan-Apochromat 63x/1.4 objective with oil immersion. The laser power was set to 3 %, using the wavelength of 488 nm and the 505 longpass filter. A region of interest was defined for a single cell and a time series was adjusted with a scan speed which allows scanning one frame within 4 s. The measurement was started and 30 µl of pNPY solution (1 µM in loading buffer) were added to the cell chamber.
4.3.2.2 Luminescence detection with CCD camera
CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells were prepared as described in section 4.2.2.2.
A 4-fold concentrated dilution series of pNPY was prepared in loading buffer and 50 µl of each concentration were pipetted into a black, flat bottomed Cellstar 96-well plate (Greiner bio-one, Solingen, Germany). 150 µl of the cell suspension were added per well at once per row with a multichannel pipette and the plate was immediately transferred into a darkbox. Recording was started with a Hamamatsu 1394 ORCA-II BTA 512 cooled CCD camera; settings were: gain: 2, exposure: 60 s, binning: 4 (16 bit). Total light dose was calculated with SimplePCI® software.
4.3.3 Results
4.3.3.1 Confocal microscopy
The calcium response could be visualized on the single cell level with confocal microscopy. Cells were loaded with fluo-4 and the signal was induced by the addition of 100 nM pNPY. Prior to the experiment, a single cell was scanned permanently for 2 min with the same laser power in order to estimate the effect of photobleaching.
During this scanning process no distinct decrease in fluorescence was observed (data not shown).
As shown in Fig. 69 addition of 100 nM pNPY to the adherent growing cell results in a steep increase in fluorescence, reaching its maximum after 8 s followed by a slow decrease in fluorescence intensity over one minute. The kinetics differs slightly from the one obtained with the flow cytometer as shown in Fig. 37 due to relevant differences between the two methods. Dye-loading, postincubation, injection of agonist (no stirring) and, most important, long measuring time (4 s per image) of a single cell and therefore low temporal resolution result in an imprecise description of the kinetic course using the confocal microscope. In addition, as shown in Fig. 37, there is a high variation in fluorescence between different individual cells, which can not be displayed by confocal microscopy. High consumable costs, the inconvenient handling especially during the addition of the agonist and the very sophisticated quantification of calcium responses account for the inferiority of confocal microscopy compared to flow cytometry.
4.3.3.2 CCD camera
As many instruments used for high throughput screening are equipped with a CCD camera, it was investigated if the light intensity generated in the aequorin assay is sufficient for quantitative measurements with a CCD camera.
Fig. 69: Time series of CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells loaded with fluo-4 in response to 100 nM pNPY measured with confocal microscopy.
Scanning time was 4 s per image. Images are shown in false colors (the warmer the color, the higher the fluorescence intensity).
As shown in Fig. 70, the luminescence light recorded for 60 s rises with increasing concentrations of the agonist. In spite of the difficult handling as described in section 4.3.2.2 the results were reproducible, and a quantitative analysis became possible.
In order to compare the results obtained from the CCD camera, the same cell preparation was used in an aequorin assay performed with the TECAN Genios Pro plate reader. The calculated EC50 values are in the same range as shown in Fig. 71.
The highest concentration of 3 µM pNPY was not used for the determination of the EC50 value with the CCD camera because the increase in luminescence starts immediately during transferring the plate into the darkbox and is therefore not completely detected by the CCD camera. This is also the reason for the higher deviations at high agonist concentrations. Nevertheless, these results show that the aequorin assay can be used for high-throughput applications using instruments equipped with a CCD camera.
Fig. 70: Overlay picture of false color presentation of light intensities generated by CHO-hY2 -K9-qi5-K9-mtAEQ-A7 cells detected with a CCD camera. Lumi-nescence signals were triggered by addition of increasing concentrations of pNPY.
Fig. 71: Comparison of concentration-response curves generated with a Hamamatsu 1394 ORCA-II BTA 512 CCD camera (panel a) and with a TECAN Genios Pro plate reader (panel b) (mean values ± SEM, n=3).
c (pNPY) [nM]
0,1 1 10 100 1000 10000
total dose
0,0 2,0e+4 4,0e+4 6,0e+4 8,0e+4 1,0e+5 1,2e+5
c (pNPY) [nM]
0,1 1 10 100 1000 10000
% of max. luminescence
0 10 20 30 40
a b
50EC50 = 55.2 ± 19.4 nM
EC50 = 71.1 ± 8.9 nM
4.4 Conclusions
The stable co-transfection with the hY2 receptor and the Gqi5 gene led to a robust calcium signal after receptor activation. The cells showed unaltered binding properties compared to the hY2-transfected cells and proved to be suitable to establish fluorescence-based functional assays. Additional stable transfection of the cells with the apoaequorin gene targeted to the mitochondrial matrix converted the calcium signal into a luminescence signal which could be quantitated by a luminescence plate reader. The pharmacological constants of receptor agonists as well as antagonists determined in the three different functional assays are in good agreement as summarized in Table 6. Slight deviations may result from different incubation periods in presence of the antagonists and different adsorption to the synthetic material of microplates, cups and other small parts used in the assays.
Table 6: Functional data of selected compounds determined in different calcium assays on hY2
receptor expressing cells.
Y2 receptor ligand Flow cytometry
(fluo-4) Spectrofluorimetry
(fura-2) Aequorin pNPY 18.1 ± 2.9a,c 16.9 ± 2.5 a,d 30.9 ± 2.2 a,d pNPY13-36 13.3 ± 6.2 a,c 18.6 ± 1.5 a,d 58.3 ± 9.9 a,d
pPYY 7.9 ± 5.5 a,c ND 8.8 ± 2.4 a,d
2 20.4 ± 2.9 b,c 28.9 ± 2.0 b,d 50.9 ± 12.9b,d
3 3.9 ± 1.2 b,c ND 69.1 ± 4.7 b,d
4 5.3 ± 0.5 b,c 14.5 ± 1.5 b,d 73.4 ± 6.1 b,d 5 101.1 ± 18.0 b,c 201.4 ± 37.3 b,d 359.4 ± 23.8 b,d 6 536.5 ± 179.5 b,c ND 521.4 ± 54.3 b,d
7 168.7 ± 62.3 b,c ND 781.5 ± 53.2 b,d
8 10.7 ± 1.1 b,c ND 50.6 ± 7.7 b,d
aEC50 [nM] of agonists; bIC50 [nM] of antagonists, calcium mobilization induced with 70 nM pNPY
cdetermined with CHO-hY2-K9-qi5-K9 cells; ddetermined with CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells
The calcium signal could be visualized by confocal microscopy as well as by a CCD camera indicating the suitability of the aequorin assay for the application in HTS-instruments equipped with a CCD camera.