Flow Cytometry Principles

Flow Cytometry is a biophysical technique used to characterize cells based on the emission of fluorescence or, in certain applications, change in impedance. Cells suspended in a stream of fluid are guided through a flow cell coupled to a detector, allowing multi-parameter analysis of up to thousands of particles per second. Although the concept of flow cytometry has rather humble roots, the technology has evolved significantly over the years to become the quintessential platform for both cell-based fluorescence quantification and cell sorting applications.

Powerful aspects of the technique stem from the ability to monitor cells in real-time, using non-destructive methods, with the potential to both characterize cell-based protein expression and allow cell-based sorting into distinct populations. As a review:

• A flow cytometer has five compartments: a flow cell, a measurement system, a detector, and amplification system, and a data analysis system.
• The flow cell uses a sheath fluid to align the cells such that they pass through the light beam and detector in single file.
• Optical measurement systems include a range of laser sources, from high-power water-cooled to low-power air cooled and diode lasers, depending on the application.
• Detectors make use of analog to digital conversion of forward-scattering, side-scattering, as well as dye-specific fluorescence signals into digital signals, that can be subsequently processed by computational methods.

In some ways, a flow cytometer is similar to a fluorescence microscope. Rather than producing a cellular image, however, the flow cytometer provides high-throughput automated quantitation of fluorescence parameters on a cell-by-cell basis. Since flow cytometry uses fluorescence quantitation as a tool, the sensitivity and real-time features are unmatched by microscopy methods -- which only collect in-focus signals from an array of individual measurements across the cell.

Modern flow cytometers possess multiple lasers and fluorescence detectors – in certain cases up to ten lasers and 30 detectors. The development of advanced instruments and a growing catalog of fluorescence probes and antibodies has fostered widespread use of the technology in both research and clinical laboratories.

Fluorescence Activated Cell Sorting

A specialized form of flow cytometry, flow activated cell sorting provides a method to sort mixed populations of cells into two distinct bins based on the fluorescence characteristics of each cell type. The physical separation of cells based on fluorescence is accomplished through an elaborate step-wise mechanism. The system first aligns the cells and detects cell fluorescence, it then disperses the cells into discrete droplets and diverts cell sub-types for collection based on an electrostatic deflection system. FACS is an acronym that was trademarked by Becton Dickinson.

Here is an excellent resource for methods, troubleshooting, and other flow cytometry fundamentals.

Flow Cytometry Applications

Flow cytometry draws popularity from the wide array of cells that can be analyzed and the incredibly diverse collection of fluorescence emitting antibodies and probes that can be used. Here are just a few applications:

• Surface exposed epitopes, antibodies, and proteins can be analyzed directly on cells through the use of fluorophore conjugated primary antibodies – a technique termed direct staining. This is particularly useful in analysis of biofluids or blood that contains target immune cells and factors.
• Indirect staining makes use of a fluorophore conjugated secondary antibody to detect a primary antibody bound to the target. This technique is useful when a conjugated primary antibody is not available and/or the fluorescence emission signal requires amplification.
• Another technique involves intracellular staining of fixed or permeabilized cells in order to detect intracellular targets. Direct or indirect staining can be employed with relevant sample preparation methods.
• Still other techniques can target DNA for cell cycle research, or can assist in harvesting native cells for staining or cell sorting. There are a wide array of flow cytometry methods and research applications which make the technology valuable across a number of fields.

Flow Cytometry Instruments

Just as there are many available reagents and seemingly endless applications, there are diverse instrument platforms and new technologies to suit. Here are a few examples of new technologies:

Beckman Coulter Gallios Flow Cytometer Research System

• The Gallios™ Flow Cytometer research system delivers analytical excellence by coupling high sensitivity, resolution, and dynamic range with high-speed data collection.
• Along with the superior detection capabilities of the instrument, the Gallios includes easy-to-use software and automation to facilitate performance of multi-color flow cytometry assays.
• With advanced optical design and sensitivity for multi-color assays, the Gallios is designed to dramatically improve user workflow.
• Four scalable formats permit versatility in laser and fluorescence detector applications, thereby expanding scope of use. Up to four solid-state lasers are available for a range of multi-color applications.
• Six detectors acquire up to six fluorescence signals, or more depending on concurrent reading needs.

View Beckman listings and get more information on the Gallios at LabX.com

Cytek Aurora Flow Cytometer

• Cytek flow cytometry solutions set new standards for flexibility, deliver excellent sensitivity, and easily resolve rare or dim populations – all at an affordable price.
• The Aurora makes use of three lasers, three scattering channels, and up to 48 fluorescence channels, providing solutions for a wide range of simple to complex applications.
• An innovative optical design provides unparalleled flexibility, enabling the use of a wide array of new fluorophore combinations without reconfiguring the system for each application.
• The optics and state-of-the-art low-noise electronics provide excellent sensitivity and resolution.
• Flat-Top beam profiles, combined with a uniquely designed fluidic system, translate to outstanding performance at high sample flow rates.
• The new SpectroFlo™ software offers an intuitive workflow from QC to data analysis with technology-enabling tools that simplify running any application.

View Cytek listings and get more information on the Aurora at LabX.com

Sysmax CyFlow® Cube 8/Cube8 Max Flow Cytometry

• The CyFlow Cube 8 is a compact flow cytometry analyzer offering modular configurations customizable for diverse research needs. This includes upgrade options for optical parameters and fluorescence channels, additional laser light sources selectable from a wide range of nine excitation wavelengths (355 - 785 nm), optional CyFlow Sorter and CyFlow Robby 8 Autoloading Station for well plates and sample tubes.
• With its small dimensions the multi-laser, CyFlow Cube 8 features built-in Windows PC, 19" LCD screen (additional second screen support), software-controlled pressure regulators, and integrated sheath/waste container. The CyFlow Cube 8 is equipped with standard interfaces for USB, LAN, video output, etc.
• The CyFlow Cube 6 Max and the CyFlow Cube 8 Max include the Blue 488 nm and the new, powerful Red 640 nm lasers. They are also enhanced with upgraded optical filters. The CyFlow Cube Max Series cytometers are also fully loaded with the upgraded CyFlow software. Easy-to-use with powerful results.

View Sysmax listings and get more information on the Cube 8 / Cube Max at LabX.com

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