Multiplexing delivers more

Dr Andrea Krumm discusses how to monitor multiple cellular reactions in a single well 

Multiplex experiments measure several biological processes simultaneously and save processing time as well as reagent costs.

The parameters of interest are measured in the same sample instead of different samples that only have a probability of being exposed to the same conditions.

In microplates, duplex-experiments that analyse two parameters are the most abundant multiplexing experiments, despite the presence of assays that analyse up to five analytes.

Analytes that are combined in a measurement are well- chosen. Many duplex assays analyse the biological process of interest and at the same time acquire a reference parameter. This is used in dual-luciferase reporter assays that relate the expression of one gene to the expression of a control gene.

Further duplex-assays analyse two biological processes in response to one experimental condition. This is applied to screen novel drugs for mitochondrial impairment. To this end, measurement of the oxygen consumption rate to report on cellular respiration is combined with extracellular acidification measurement to reflect glycolytic activity.

Two parameters may also be required to unambiguously describe one biological process. For instance, calcium release is not sufficient to report Gq -type receptor activation.

A second messenger generated upon Gq stimulation such as diacylglycerol (DAG) helps to identify Gq-activation. 

Technical considerations

The measurement of two or more analytes is achieved by two methods. One solution combines different detection modes such as luminescence and absorbance.

The second option employs spectrally resolved fluorophores, for instance a fluorophore that emits in the green with one that emits in the red range of light. Microplate readers that are intended to detect multiplex assays are ideally monochromator-based multi-mode readers to provide the highest flexibility in terms of detection modes and fluorophores.

The Clariostar microplate reader by BMG Labtech not only meets these criteria, but also increases the sensitivity and ease of use of multiplexed measurements.

The monochromator is based on linear variable filters (LVF) that have variable coatings along their lengths that reject or pass certain wavelengths of light (Fig. 1).

The LVF monochromatorT is advantageous for multiplexing since wavelengths and bandwidths can be individually adjusted to guarantee best separation of the fluorophores or luminescence signals. In comparison to grating-based monochromators, the LVF monochromator provides higher light transmission, which can be further increased by extension of bandwidths to 100nm. This way assay sensitivity is improved.

Another important factor for the detection of two fluorophores is the use of a dichroic mirror that reflects background light at lower wavelengths than light arising from autofluorescence, excitation light or another fluorophore.

The Clariostar uses a variable dichroic that is automatically adjusted to separate the actual emission from interfering light.

Apart from spectral separation of analytes without losing sensitivity, the detecting device should support general advances of assay development. Cell-based assays are on the rise to be closer to physiological settings. To avoid measuring fluorescence of medium and particles therein it is suggested to detect fluorescence of adherent cells from the bottom of a well.

Furthermore, kinetic measurements are often preferred over endpoint analysis to guarantee to acquire data at the time-point of reaction. The kinetic measurements need to be managed by the microplate reader, irrespective of fast or slow reactions.

Simultaneous measurements

Because it offers these features, the Clariostar microplate reader has been employed to validate novel fluorescent biosensors for second messengers. It shows the combination of a red emitting calcium indicator with a green fluorescent DAG-sensor to identify stimulators of Gq-type GPCRs. Both fluorophores can be genetically encoded in cells relevant to the disease and increase their fluorescence intensity with increasing analyte concentrations.

The sensors achieve optimal expression in mammalian cells due to packaging in a modified baculovirus (BacMam). The DAG-sensor has a circularly permuted fluorescent protein near the DAG binding domain of protein kinase C. Upon DAG binding, the sensor undergoes  a conformational change that changes fluorescence intensity.

Carbachol was used to stimulate the Gq -coupled hM1 receptor in HEK293 cells and to induce Gq relevant second messengers.

Upon injection of the compound, the red fluorescent calcium indicator R-GECO reported a rapid calcium release and subsequent drop of cytoplasmic calcium (Fig. 2).

Simultaneously, a green fluorescent DAG-sensor was measured that detected a fast production of DAG that decreased more slowly. Two minutes after stimulation the DAG level was at 60% of the maximal content just after injection.

The development of spectrally resolved fluorophores and biosensors along with the advances in microplate readers extend the output of a biological experiment. In one experiment it is now possible to monitor the time-response of several cellular reactions.

Dr Andrea Krumm is application specialist at BMG Labtech

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