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Fluorescence-Activated Cell Sorter

Nearly 35 years since Stanford researcher Leonard Herzenberg and colleagues developed the first fluorescence activated cell sorter (FACS), the instrument has become the immunologists' key tool. Immunology journals are chock-full of flow-cytometry profiles, the characteristic plots that such instruments produce.But cytometry is just half the story. The instruments also allow researchers to purify specific cell populations based on the presence or absence of particular characteristics. And therein

By | July 19, 2004

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Nearly 35 years since Stanford researcher Leonard Herzenberg and colleagues developed the first fluorescence activated cell sorter (FACS), the instrument has become the immunologists' key tool. Immunology journals are chock-full of flow-cytometry profiles, the characteristic plots that such instruments produce.

But cytometry is just half the story. The instruments also allow researchers to purify specific cell populations based on the presence or absence of particular characteristics. And therein lies their value. Using a FACS, a scientist can isolate and purify rare dendritic cells or hematopoietic stem cells from a patient's blood, for instance.

Herzenberg's prototypical FACS used a mercury arc light source and could measure just one parameter, fluorescence intensity. With monoclonal antibodies only just discovered, there were scant markers to study in any event. Data were archived by photographing an oscilloscope screen, and the instrument could process a mere 100,000 cells per minute.

Today's immunologists, in contrast, have access to instruments capable of sorting several million cells per minute, with multiple lasers, advanced computer and software support, thousands of monoclonal antibodies, and dozens of fluorophores from which to choose.

BD Biosciences-Immunocytometry Systems, a division of Becton-Dickinson, which licensed Stanford's FACS patents, recently rolled out its newest instrument, the BD FACSAria. Its key components are diagrammed below.

1 Light from the BD FACSAria's three lasers (407, 488, and 633 nm) are delivered via fiber optic cables to alignment prisms, which focus the light onto the cuvette flow cell.

2 In the flowcell, the cells, tagged with a cocktail of fluorescently labeled antibodies and intracellular dyes, are hydrodynamically focused by injection into a laminar flow of "sheath fluid," which keeps the cells separated and in a single file.

3 As cells pass single-file through the cuvette, flowing at 70,000 to more than 100,000 cells per second, they are interrogated for the presence or absence of fluorescent markers. The cells are then dispersed into unicellular droplets, given an electric charge based on the fluorescent data, and guided into collection chambers by two charged deflection plates; uncharged particles go to the waste bin. The BD FACSAria supports four-way sorting, with yield exceeding 80% and purity exceeding 98%.

4 Each laser's signal is processed separately. An octagon-shaped collection device collects the signal from the 488-nm laser. A series of long-pass dichroic mirrors allows wavelengths of interest to pass through to a detector, while reflecting shorter wavelengths to the next detector. Behind each mirror a bandpass filter further restricts the incoming light to a wavelength of interest. In this way, the detection array reads seven fluorescent channels plus side scatter (a measure of cell granularity) from the single laser. Similar trigon-shaped devices detect three colors each from the 633- and 407-nm lasers. With the addition of forward scatter (which measures cell size), the system can thus detect up to 15 parameters simultaneously.

Jeffrey M. Perkel can be contacted at jperkel@the-scientist.com.

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