Biomedical Engineering Reference
In-Depth Information
Hertzenberg from Stanford University. The first simple instrument could
separate cells based on cell size, granularity, and one or two “colors” us-
ing different fluorescent tags. Today, with the advent of new fluorescent
probes, three different fluorescent dyes can be used with a single laser
(after the “compensation” is corrected for signal spillover between chan-
nels). FACS instruments are now routinely fitted with two lasers and are
designed to separate cells based on eight or nine individual parameters
using fluors with distinct excitation and emissions spectra. There are two
functions of cell sorters, one to analyze subpopulations of cells, and the
other to collect these different populations. The FACS is routinely used
not only for basic scientific analysis in immunology and cell biology, but
is also used for analysis of clinical specimens and for screening of novel
monoclonal (including humanized) antibodies against various tissues,
together with a host of other uses.
The FACS instrument basically has two types of detectors. The light
detector is used to characterize cells that enter the analyzers by for-
ward and side scatter. Forward and side scatter are used to discriminate
between different types of cell populations in a mixed sample. Forward
scatter provides information that can be used to determine the rela-
tive size of cells that are being analyzed through a FACS instrument.
Side scatter provides information about the granularity of a population.
Thus, for example, if white blood cells from the blood or spleen are
analyzed through a FACS instrument, forward and side scatter can be
used to identify the lymphocyte, monocyte, and macrophage popula-
tions and discriminate them from neutrophils and other granulocytes. In
addition, photomultiplier tubes collect information based on the fluores-
cence emissions spectra of the cells that are being passaged through
the instrument to determine what types of antibodies (and therefore
what types of surface molecules) are present on individual cell pop-
ulations. The excitation of the fluorescent tags is accomplished using
one or more lasers that are capable of exciting fluors at different wave-
lengths.
Fluorescence labeling allows the identification of cells or organelles
with different cell surface molecules. The FACS instrument can collect
fluorescent signals in one of a number of different “channels” that corre-
spond to different laser excitation and fluorescent emission wavelength.
Cells can be analyzed at rates of 100,000 to 1,000,000 cells per minute
(slower flow rates are generally required when sorting cells, see below).
Fluorescence can be detected using antibodies in one of two ways.
First, an antibody against a cell surface molecule can be conjugated
with a fluorescent tag (direct immunofluorescence). Alternatively, an an-
tibody against a cell surface marker can be utilized in an unconjugated
form and detected using a secondary antibody, a method called indirect
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