( L. Smith1 M.J. Price-Jones 1...)Counting Cytostar-T scintillating microplates on the MicroBeta JET

L. Smith1, M.J. Price-Jones¹, K.T. Hughes¹ and N.R.A. Jones² (¹ Nycomed Amersham plc., Forest Farm, Whitchurch, Cardiff, CF4 7YT, Wales, UK, ² PerkinElmer Life Sciences, P.O.Box 10, FIN-20101, Turku, Finland)

INTRODUCTION

MicroBeta JET is a multi-detector instrument designed for liquid scintillation (LS) or luminescence detection of samples in microtitration plates, tubes or on filters.

Three different MicroBeta models are available. Two manually loaded models have sample capacities of up to 16 or 32 plates. The third model is fitted with an external loading platform for fully automatic loading by robot systems.

MicroBeta JET has either 1, 2, 3 or 6 detectors and can count in either 96 or 24 formats. Each MicroBeta detector comprises two photo-multiplier tubes (PMTs), one above the sample and one below. This arrangement provides the best possible counting geometry for every assay type.

For routine LSC, coincidence counting is a robust and sensitive method. Coincidence counting is independent of the type of scintillator and is unaffected by changes in temperature. In an ideal sample tritium efficiency is in excess of 55% whilst the background is typically less than 10 CPM.

In the case of measurement of Scintillation Proximity Assay (SPA) or Cytostar-T scintillating microplates ParaLux Count Mode is recommended because this maximizes the advantage of the lower PMT. In these assays the radio-label is much closer to the lower PMT and as a consequence considerably higher counting efficiencies can be obtained. ParaLux has also been shown to give superior color quench correction¹.

The novel feature in JET is the inclusion of multiple reagent injectors for measuring prompt (or flash) reactions. Multi-detector JETS can have one or two Injector Modules installed for one or two injection and counting sequences. The single detector JET can have up to four Injector Modules.

Multi-detector JETS can be set to deliver different reagents to the reading position of each detector and therefore specific wells in each plate. For example, in the case of a six detector JET fitted with one injector module, up to six different reagents can be dispensed across each plate.

Counting control includes the facility for repeat measurements of each sample after injection. In this fashion kinetic analysis of reactions lasting seconds or longer can be studied.

In terms of instrument design, if reagent injection is selected in any particular counting protocol, an Injector Assembly is automatically positioned between the sample plate and the upper PM tubes. This injector assembly houses the reagent tubes and fiber optic light guides. If reagent injection is not necessary, performance of the MicroBeta JET is identical to the MicroBeta TriLux.

Assays based on Amersham Pharmacia Biotechs Cytostar-T scintillating microplates are highly suitable for MicroBeta JET. This plate allows the real time analysis of a broad spectrum of cell associated phenomena to be studied over a defined period of time.

EXPERIMENTAL

Cell based assays were set up using Cytostar- T scintillating microplates to determine the effect of selective agonists and antagonists on cloned GluR6 receptor channels expressed in BHK cells. Glutamate gated ion channels are permeable to cations such as Na⁺, K⁺ and Ca²⁺, therefore allowing [¹⁴C] guanidinium (a marker for Na⁺ ions) to be used in ion flux experiments. The use of the sodium channel blocker, procaine, allows the influx of [¹⁴C] guanidinium into the cells through the GluR6 receptor channel when it is stimulated with selective agonists such as kainate and domoate.

BHK cells (supplied by Novo Nordisk) transfected with the inducible GluR6 ion channel were grown as monolayers in 75 cm² flasks at 37 C in a humidified 5% CO₂ incubator. GluR6 expression was induced in the cells with 5 mM (final concentration) isopropyl- 1-thio-β-D-galactopyranoside (IPTG). All dilutions of the agonist, antagonist and [¹⁴C] guanidinium (from Amersham Pharmacia Biotech) were prepared in assay buffer, consisting of NMG buffer containing 10 mM HEPES, pH 7.5, 2 mM CaCl₂, 150 mM Nmethyl- D-glucamine and Ringers buffer containing 10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM KCl, 1 mM CaCl₂ and 20 mM sucrose. 4 parts NMG and 1 part Ringers buffer were prepared containing an additional 2.2 mM CaCl₂, to which was added 500 μM procaine and 1 mg/ml concanavalin A, to prevent receptor desensitization.

ADDITION OF THE AGONIST DOMOATE USING THE MICROBETA JET

Assays were set up to compare the effect of adding the agonist manually to the wells of Cytostar-T microplates and adding the agonist through the injectors of the MicroBeta JET. Duplicate wells were assayed containing 50 μl [¹⁴C] guanidinium (10 μCi/ml) to which was added 50 μl of the prepared agonist. No agonist control wells were also assayed. These contained assay buffer instead of the agonist. After all additions were completed, replicate wells were counted simultaneously on different detectors for 20 secs/well using repeated counting cycles.

ADDITION OF THE ANTAGONIST KYNURENIC ACID USING THE MICROBETA JET

The effect of the antagonist kynurenic acid was detrmined on the GluR6 assay system. Duplicate wells were assayed containing 50 μl [¹⁴C] guanidinium and either 0.1 μM domoate or 0.1 μM domoate/200 μM kynurenic acid. The 0.1 μM domoate/ 200 μM kynurenic acid was added to the wells as one solution and the agonist induced response measured by counting Cytostar-T microplates for 20 secs/well over 10 cycles.

RESULTS

Figures 1 and 2 demonstrate the advantage of using the MicroBeta JET to add the agonist to the wells of Cytostar-T scintillating microplates. Using the MicroBeta JET, the induced response can be followed immediately after addition of the agonist. Adding the agonist manually results in a lag time where the initial response to the agonist cannot be measured, giving a significant difference in counts obtained for domoate in the first counting cycle. The lag time can be overcome using the MicroBeta JET to perform additions of the agonist to the wells of the Cytostar-T scintillating microplates.

The immediate effect of adding the antagonist kynurenic acid is shown in Figure 3. Suppression of the agonist induced response was immediately seen on addition of the domoate/kynurenic acid solution to the wells of Cytostar-T scintillating microplates. This was shown as a decrease in signal which was dependent on the concentrations of agonist and antagonist used in the assay.

Manual addition of kynurenic acid would give a similar delay in the initial response, as shown in Figure 2, resulting in a significant proportion of the antagonist effect of kynurenic acid being lost in that time.

CONCLUSION

Addition of reagents to the wells of the Cytostar-T microplate using the MicroBeta JET allows the response generated by addition of both agonist and antagonist to be measured immediately. Previously, on addition of these reagents manually, it was not feasible to obtain several measurements within a 5 minute time period, which resulted in a lag time where it was not possible to determine the immediate effect of the agonist or antagonist on the cells. This can now be overcome using The MicroBeta JET.

REFERENCES

1. Application Note: Color quench correction in Scintillation Proximity Assays using ParaLux Count Mode. 1450-1024.

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