This page contains a Flash digital edition of a book.
MICROPLATE READERS 29


Multiplexing delivers more M


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


ultiplex experiments measure several biological processes


simultaneously and save processing time as well as reagent costs. Te 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. Tis 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. Tis 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 Te measurement of two or more analytes is achieved by two methods. One solution


combines different detection modes such as luminescence and absorbance. Te 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.


Te Clariostar microplate


reader by BMG Labtech not only meets these criteria, but also increases the sensitivity and ease of use of multiplexed measurements. Te 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). Te LVF monochromator is


www.scientistlive.com


Fig. 1. A linear variable long pass filter slide (shown) is one of the specialised components that make up the Clariostar’s sophisticated LVF monochromator system


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76