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Techniques Series: Using Flow Cytometry

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Flow Cytometry is a powerful technique that enables researchers to conduct a rapid and multi-parametric analysis of single cells simultaneously.

Of late, it has become an indispensable tool in basic cell biology and medical research, immunological studies, drug discovery, and even diagnosis of diseases. The applications vary as well, from cell sorting of heterogeneous populations, DNA abnormalities, apoptosis assays, cell cycle analyses, immunophenotyping, protein modifications, proliferation assays, and cell signaling.

Below is a broad overview of the technique platform, how it works in these applications, and new technological developments.

Flow Cytometry Platform

The Flow Cytometry platform is composed of four basic components, including a fluidic system, optics, electronics, and computer software.

The fluidic system contains a sheath fluid to keep the cells to be analyzed suspended, and allow for alignment of a single file of cells as it passes through an optics system. The optics system is comprised of a laser diode that provides the source of light. The system also includes lenses that can focus the laser beam on cells, and optical mirrors and filters that help to route scattered light or fluorescence into appropriate detectors.

The electronics components convert the data of the detectors, amplify them, and convert them into electrical signals or voltage. Computer software than digitally converts these signals, storing the data in a Flow Cytometry Standard (FCS) format, and permitting for analysis conducted with any accompanying software.

How Flow Cytometry Works

The 4-component based Flow Cytometry system can in turn exploits varying optical and fluorescent characteristics of single cells.

With the fluidic system used to analyze a cell suspension, and hydro-dynamically focused, single cells may pass rapidly in front of a focused-laser diode. The cells scatter the light that is collected by the detectors: one placed in line with the laser, which computes a cell’s relative size (Forward scatter) and others placed at right angles to the laser path, which determine a cell’s interior complexity (e.g. cell granularity, organelles etc.) or fluorescence (Side Scatter).

Flow Cytometry also allows for specific protein antigens of cells, on surface or within it, to be detected using specific antibodies conjugated to fluorescent dyes. In this case, the laser excites the fluorescent molecules to a higher energy state, and the emission of light energy at different wavelengths allows several parameters of cells to be analyzed simultaneously.

Lastly, scattered light signals are passed through specific filters and collected by appropriate detectors. The signals are then amplified and converted into electrical signals (voltage) to be analyzed by a computer.

Applications & New Technologies

One of the most common and important applications of using such a platform is to study expression of protein antigens of cells. During my Ph.D., I routinely used Flow Cytometry to analyze the expression of receptor tyrosine kinases (RTK) on surface and within the cells (Read my paper).

As technology continues to improve though, new generation of flow cytometers will emerge with additional applications. A flow cytometer analyzer by BD sciences for instance has up to 5 lasers and allows for studying of 20 parameters simultaneously. Such a flow cytometer would be critical to analyze properties of extremely rare cell populations and lead to important medical and basic science discoveries.

One of the caveats of standard Flow Cytometry is the lack of information about the localization of protein antigens and antigen-interactions. The advent of new generation of flow cytomers such as Amnis imaging flow cytometers, which combine power of digital microscopy and Flow Cytometry, has made it possible to visualize localization of antigens while also gathering its expression level at the same time. Such simultaneous acquisition of quantitative and qualitative data will greatly increase the scope and efficiency of research.

If you want to order Flow Cytometry for your own study, check out the 33 facilities who offer the service on Science Exchange: https://scienceexchange.com/services/flow-cytometry

 

[about_box image=”http://thebenchapp.s3.amazonaws.com/wp-content/uploads/2012/05/Roshan.jpeg”]Roshan Karki is a Postdoctoral Research Associate at Danbury Hospital (part of the Western Connecticut Health Network). His research is focused on developing biomarkers for gynecological cancers. He is also a member of the Science Advocate program and believes Science Exchange has the potential to impact scientific research and facilitate medical and drug discoveries. Previously, Roshan completed a Ph.D. in Experimental Pathology at Yale University. [/about_box] 

 

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Roshan Karki

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