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Flow Cytometry


Figure 1


The Bio-Rad Laboratories ZE5 Cell Analyzer is capable of


detecting up to 100,000 events every second


cells with specific phenotypic features generate dif- fering emission spectra, valuable cell characterisa- tion information can be rapidly obtained. Ongoing advances in instrument design have


resulted in the commercial availability of flow cytometers with an extensive range of laser and detector options. Together with improvements in the range of fluorescent dyes available for cell stain- ing, this has led to an expansion in the number of wavelengths or ‘channels’ that can be used to probe specific cell parameters. While these developments have enhanced the analytical power of multichan- nel flow cytometry as a screening technique, this also necessitates the careful selection of laser, fluo- rochrome and detector technologies to maximise the amount of useful information generated. One of the most important factors affecting the


quality of flow cytometry data is the choice of channels used to measure specific cell parameters. However, this task is complicated by the fact that some chromophores fluoresce at very similar wave- lengths, with varying degrees of intensity. If a detec- tor assigned to pick up signals from a fluorochrome of interest detects fluorescence emission from a sec- ond fluorochrome, the physical overlap of emission spectra (known as spectral spillover) can make data interpretation challenging. This issue is even more acute if the fluorochrome of interest is weakly-emit- ting or dim, making the effects of interference even more difficult to deconvolute. To address this issue, many instrument and reagent vendors are expand- ing and enhancing the range of detection channels


32


available for flow cytometry experiments. One approach is to improve the quality of fluo- rochromes used for cell staining. “The limited availability of high-quality dyes


with high extinction co-efficients has been a major problem for laboratories, leading to sub-optimal resolution of positive and negative cell popula- tions,” explains Dr Robert Balderas, Vice- President, Market Development for Biosciences at BD Biosciences. “Our polymer dye technology platform gives researchers access to a broad range of fluorochromes, many of which are 20 times brighter than traditional dyes. This not only facili- tates the use of new laser lines, it adds additional options to existing ones too.” Another strategy to enhance the quality of flow


cytometry measurements, especially when using dim fluorochromes, is to improve instrument sensi- tivity. One way this can be achieved is by using advanced detection technologies. “Some of the most exciting advances in flow


cytometry design relate to improvements in detec- tor technology,” notes Dr Nancy Li, Vice-President of Engineering at ACEA Biosciences Inc, part of Agilent Technologies. “Silicon photomultiplier (SiPM) technology, for example, offers high gain and high sensitivity for photodetection, which makes it possible to simultaneously measure a large number of fluorophores with excellent signal resolution. Our NovoCyte® Quanteon™ system is the first flow cytometer to adopt SiPMs for fluores- cence channel detection.”


Drug Discovery World Spring 2019


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