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Fig 2. Multiplexed DAG and Ca2+


kinetics. Traces depict the average response to 30 μM Carbachol


(n=16) expressed as 100% response. R-GECO (red); green DAG (green). Carbachol was dispensed using on-board reagent injectors at the 30-second time point as indicated


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. Tis 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. Te 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


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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. Te 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.


Te sensors achieve optimal


expression in mammalian cells due to packaging in a modified baculovirus (BacMam). Te


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. Te 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. www.bmglabtech.com


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