Selection of transfected cell clones

The CHO-hY2-K9-qi5-K9 cells were transfected with the Eam11051-linearized pcDNA3.1/Zeo-mtAEQ vector. 96 cell clones were loaded with 2 µM coelenterazine h and screened on the basis of their luminescence signal after cell lysis caused by 0.1% triton-X-100 (see Fig. 51). Provided that the cell density per well is similar, a strong luminescence signal should indicate high expression of functional apoaequorin. Three cell clones were selected, expanded and tested for their functional response upon agonist challenge.

As shown in Fig. 52, the particular cell clones responded differently. Each clone showed a saturable, concentration-dependent increase in luminescence upon stimulation with pNPY. The strongest signals were obtained with clones B4 and E7 (Fig. 52b) but even injection of the cells to solvent without agonist led to an increase in luminescence as shown exemplarily for clone E7 in Fig. 52a. This results in a high basal signal and therefore impairs the signal to noise ratio. The injection of cell suspension of clone A7 to solvent led only to a minimal increase in basal luminescence (Fig. 52a), resulting in a higher signal-to-noise ratio. The maximum increase in the luminescence signal upon saturating concentrations (3 µM) of pNPY was 16-fold with clone A7 compared to 9.3- and 5-fold with the clones B4 and E7 respectively. Injection of the pool of transfected cells to solvent led to a basal signals ranging from the ones of clone A7 to those of E7. With this mixture of cell clones a maximum signal to noise ratio of 11.6 was obtained. Cell clones B4 and E7 were

Fig. 52: Luminescence responses of isolated CHO-hY2-K9-qi5-K9-mtAEQ cell clones after stimulation with increasing concentrations of pNPY (n=3; mean values ± SEM).

time [s] clone E7; 300 nM pNPY clone A7; solvent clone A7; 300 nM pNPY

c (pNPY) [nM]

1 10 100 1000 10000

area

frozen and the cell line CHO-hY2-K9-qi5-K9-mtAEQ-A7 was used for the investigations on assay parameters.

4.2.3.2 Optimisation of assay parameters

In order to obtain reproducible results cells have to be prepared in a way that their response is constant during the measurement. As a single luminescence signal with these cells takes at least 40 s (see Fig. 52a), the measurement of a complete 96-well plate takes 64 min. The stability of the signal during this period is a prerequisite for the application of the assay in the 96-well format. The signal intensity depends on the concentration of reconstituted aequorin within the cells and the stimulus caused by a defined agonist concentration. As the latter remains unchanged during the measurement, parameters have to be found to provide constant concentrations of reconstituted aequorin. Due to the basal Ca²⁺ concentration within the mitochondria, aequorin is constantly discharged, but on the other hand, sufficient coelenterazine is available to reconstitute active aequorin because coelenterazine is kept continually in the cell suspension. Incubation conditions have to be found which allow a constant steady-state level of active aequorin depending on the equilibrium between entry of coelenterazine in the cell by passive diffusion, reconstitution of aequorin from apoaequorin and its consumption in the basal nonstimulated cell’s conditions. In the literature there are different coelenterazine loading conditions described. For example Stables and colleagues report on the reconstitution of the holoenzyme by incubation of adherent growing transiently transfected CHO cells with 5 µM coelenterazine for 3 h at 37 °C in culture medium (Stables et al., 1997). This

Fig. 53: Effect of postincubation time on the concentration-response curves of pNPY. CHO-hY2 -K9-qi5-K9-mtAEQ-A7 cells were incubated for 2 h with 2 µM coelenterazine h and post-incubated for 90 min (panel a) or 3 h (panel b). Total aequorin (100 %) was discharged after cell lysis by 0.1 % triton-X-100. Measurment time of each curve was 8 min (40 s per concentration).

c (pNPY) [nM]

procedure requires large amounts of the cofactor as only a small number of cells is loaded in a large volume. Incubation at 37 °C seems not to be very economic as more aequorin is consumed under basal conditions at higher temperatures (Blinks, 1978; Dupriez et al., 2002). Loading of the cells in suspension at a high cell density and subsequent dilution appeared to be an effective and economic method (Dupriez et al., 2002). After dilution with loading buffer, cells were “postincubated” at room temperature for different periods and concentration-response curves of pNPY were recorded. As shown in Fig. 53a, postincubation of 90 min is insufficient as luminescence signals increase during the measurement. Reproducible concentration-response curves were obtained after 3 h of postincubation (Fig. 53b). The data points of all curves were in the same range with acceptable deviations. This allows a temporal assay window of at least 64 min, sufficient for the measurement of a whole 96-well plate.

Another aspect of the aequorin assay is the use of different coelenterazine derivates and their employed concentrations. As the synthetic commercially available derivates

are very expensive, the coelenterazine h derivate was chosen based on published results (Dupriez et al., 2002). Using CHO-K1 cells stably coexpressing apoaequorin and the human 5-HT2B receptor in a functional aequorin assay, coelenterazine h proofed to be superior to the native and to the other synthetic derivatives coelenterazine n, f, hcp, cp and the benzyl derivative in terms of sensitivity and signal-to-noise ratio. To determine the optimal concentration of the cofactor, the cells were incubated in the presence of increasing concentrations of coelenterazine h.

Cells were diluted and postincubated for 3 h. The results are shown in

Fig. 54. There is no distinct tendency, but the strongest signals and the lowest EC50

values were obtained after loading of the cells with cofactor at a concentration of 2 µM.

c (pNPY) [nM]

1 10 100 1000

% of max. luminescence

0 10 20 30 40

6 µM coelenterazine h 4 µM coelenterazine h

2 µM coelenterazine h Fig. 54: Effect of various concentrations of coelenterazine h during the loading pro-cedure. Cells were loaded for 2 h, diluted and postincubated for 3 h at room tem-perature (mean values ± SEM, n=3).

As test compounds are often dissolved in DMSO, the sensibility of the assay depending on the DMSO content was investigated. Cells were loaded with 2 µM coelenterazine h and postincubated with increasing DMSO concentrations for 3 h.

Concentration-response curves of the agonist pNPY are shown in Fig. 55. Increasing concentrations of DMSO led to an elevated basal signal. This effect is presumably due to the impairment of the cells by the solvent. At 1 % of DMSO there is still a distinct concentration-dependent increase in emitted light after stimulation with pNPY (Fig. 55a). The EC50 value is in the same range (147.9 nM) as that of control cells, which were postincubated in absence of solvent (EC50 = 165.7 nM). Calculation of the percentage of maximal luminescence using the signal of cells lysed by 0.1 % triton-X-100 (100 % value) results in concentration-response curves almost identical for control cells and those incubated with 1 % DMSO (Fig. 55b). A reason for this is

the higher basal consumption of aequorin in unstimulated cells in the presence of DMSO, which diminishes the 100 % value (triton-X-100) and increases the 0 % value.

Incubation with 2 % and 5 % solvent caused considerably higher basal signals and greater deviations.

The maximally emitted light (100% value, not shown) of cells incubated with 5 % DMSO is only 45 % of the maximal signal obtained with the control cells, whereas the blank signal was 2.3-fold as high as the blank signal of the control cells.

As a final concentration of 1 % DMSO only marginally affected the concentration-response curves, this concentration can be used in the screening of antagonists

c (pNPY) [nM]

1 10 100 1000 10000

% of max. luminescence

Fig. 55: Effect of DMSO. CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells were loaded with 2 µM coelenterazine h for 2 h, diluted and postincubated for 3 h with indicated DMSO contents. In panel a total emitted light is calculated from peak integrations of each signal. In panel b the percentage of maximal luminescence is calculated with the 100 % triton-X-100 signal as standard (mean values ± SEM, n=3).

b

c (pNPY) [nM]

1 10 100 1000 10000

emitted light [RLU]

dissolved in DMSO. Additionally, in the assay, the antagonists dissolved in DMSO are preincubated with the cells for 1 h (see section 4.2.3.4) instead of 3 h, which diminishes the effect of the solvent.