Quantifying antibodies with flow cytometry

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by: Gen Federico
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Word Count: 346
Date: Thu, 17 Nov 2011 Time: 6:29 AM

For each target protein listed on our antibody database, we at Novus Biologicals aim to offer a maximum choice of antibodies, allowing you to customize your research. This includes antibodies bound (conjugated) to fluorescent dyes, or fluorophores, for use in fluorescence microscopy, flow cytometry and similar analysis.

Flow cytometry utilizes a beam of light tuned to a specific wavelength, which is focused onto a stream of fluid containing particles or cells in suspension. A number of detectors are stationed at the point where the fluid passes through the laser beam; one in line for forward scatter (FSc) and several more at a perpendicular angle for side scatter (SSc). One or more separate detectors also measure fluorescence, one for each emission peak.

FC works on a dual principle: the first, that any particle of 0.2 to 150 micrometers in size will scatter the light in some way; and the second, that fluorescent dyes bound to the particles and excited at a specific wavelength will emit light at another specific wavelength. An ADC (analog-to-digital conversion) system translates the FSC, SSC and fluorescence information into electrical signals for digital interpretation by computer.

Modern flow cytometers can analyze several thousand particles each second, and generally have multiple lasers and detectors for multiple labeling of antibodies, allowing more precise identification of cellular markers. By measuring scatter and fluorescence fluctuations at various emission peaks, an enormous amount of information is derived. SSc gives us information on cellular complexity and/or granularity; FSc tells us the cellular size, while fluorescent antibody labeling allows us to study the structure and function of the cell. FACS, or Fluorescence Activated Cell Sorting, takes the principle one stage further, allowing cell populations to be isolated and separated according to their physical characteristics and fluorescence levels.

Antibody suppliers now have an almost limitless number of fluors with which to conjugate antibodies, over the entire spectrum from near-infra red to ultraviolet wavelengths. Our antibody catalog includes Alexa, Texas Red, Fluorescein; FITC; HiLyte and DyLight Fluors, covering every excitation and emission wavelength.

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