Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening ( n is then recorded usually for 20 secon...)

Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening

n is then recorded usually for 20 seconds. By injecting the same cell suspension into each of the 96 wells, this method avoids the need to wash the dispenser(s) between each measurement and allows 96 measurements of agonist-induced aequorin light emission in 32 minutes with a single-dispenser luminometer. However, a six-detector MicroBeta JET can perform 96 measurements in six minutes or up to 10,000 measurements in one day.

Once the cells have been incubated with coelenterazine, they can be used for several hours and even several days for the measurement of agonist-induced increase of intracellular calcium concentration. A signal-tonoise ratio above 50 was currently obtained with this system of cell injection.

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COST OF THE METHOD

The assay costs are minimal. In a typical experiment, 50 x 10⁶ cells are resuspended in 10 ml of buffer and coelenterazine is added at a final concentration of 5 μM. After reconstitution of active aequorin, the cells are diluted 10 times before use, and 50 μl of the dilution (i.e. 25 000 cells) are injected per well. The coelenterazine loading of cells in suspension and at high density avoids the big consumption of coelenterazine that would be necessary if the cells were loaded while attached to the plates. This renders the method very cheap. The cost of coelenterazine is only 0.014 US$/well (approximately 0.015 Euros or 0.025 DM). The volume of the cell suspension necessary to fill the dead volume of the injectors of the luminometer is usually between 1.5 mL (single injector devices) and 6 ml (6 detector MicroBeta JET); thus it costs between 0.4 and 1.70 US$ to fill the dead volume.

Plates: Either solid white or white-side / clear bottom 96-well plates can be used. The approximate cost per well is 0.04 US$/well

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