INTRODUCTION TO FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 3

early flow cytometers since reagents were available that not only bound specifically to
DNA (e.g., ethidium bromide developed by Dittrich and Gohde in 1969 [49]) but also
emitted fluorescence when excited with a laser. Much of the essentials of the modern-
day FACS are the same as those in the early flow cytometers. However, these early
flow cytometers were cumbersome and required an on-site engineer. The laser was
water cooled and alignment issues were critical. In addition, no computer was
attached to these early flow cytometers, nor were programs available for data
analysis [50]. At one point, we took Polaroid pictures of oscilloscopes and sent data
to a DEC10 supercomputer and wrote our own programs for cell cycle analysis.
Although the development of FACS depended on many advances in various
disciplines including dye chemistry, electronics, and computers, one important
breakthrough that was critical for the development of flow cytometers was the
principle of measuring cells or particles in liquid suspension. Advances in the flow
principle began in 1940withCrosland-Taylor using the flowprinciple and light scatter
to measure blood cells [51]. The breakthrough technology was first developed by
Coulter and the Coulter principle describes changes in the electrical conductivity of a
small saline-filled orifice as a cell passes through it. In 1953, Wallace Coulter and his
brother Joe obtained a U.S. patent for the Coulter counter that automated counting of
particles, particularly cells in the blood [52]. The use of a liquid stream (or a sheath) to
which a sample is introduced allows individual cells to be distributed in the sheath that
then passes through a nozzle (detecting electrical conductivity changes) to generate a
trigger, which indicates the presence of a signal that exceeds the threshold level.
Many of the applications for FACS analysis involve the identification of membrane
markers via the use of fluorochrome-tagged antibodies, which recognize these
markers. Many of these membrane markers are surface proteins or surface antigens,
which help to define the cell. These antigens are used to classify the cells and are often
assigned a cluster of differentiation number or a CD number. Antibodies (which are
normally produced by B lymphocytes) can be made that specifically bind to these CD
molecules. There are more than 200 CD molecules that have been identified and
specific antibodies have been produced that recognized these CDmarkers [53-55]. In
addition, many of these antibodies are commercially available as labeled antibodies
with different fluorochromes.
1.2 BASIC PRINCIPLES OF HOW A FLOW CYTOMETER WORKS
The basic components of a flow cytometer (Figure 1.2) consist of (1) a flow cell that
forces single cells into the middle of a fluidic sheath, (2) a laser source of light, (3)
optical components to focus light of different wavelengths (colors) onto a detector, (4)
a photomultiplier to amplify the signal, and (5) a computer.
In a basic flow cytometer, the sample (containing the cells tagged with fluor-
ochromes in a liquid) is drawn up and pumped into the flow cell through tubing. The
cells flow through the flow chamber rapidly and singly and are passed through one or
more laser light beams. As the laser beam hits the cells, the light beam is scattered in a
forward direction and a side direction. Fluorescence emission can also be detected.