Instrumentation
FACSVantage with TurboSort: This flow cytometer is capable of
high speed cell interrogation and sorting. The electronics of the instrument
have recently been redesigned for pulse process speeds in excess of 2 MHz.
This increased speed is reflected in viable cell recoveries in excess of
70% at sorting speeds of 20K/sec. This instrument has other modifications
to provide advanced flow cytometry applications.
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Forward Scatter Detectors: These include both forward
angle scatter and forward angle fluorescence detection (FFD). FFD is a
new parameter that may be useful in providing additional cell resolving
power.
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Seven color detection: Standard flow cytometry instrumentation
provides up to nine measurement parameter usually consisting of FSC, SSC
and seven colors of fluorescence. Excitation is from three spatially separated
lasers (UV, 488, 560 - 640 nM tunable dye lasers) with three independent
timing windows. Using the primary beam for 488 nM excitation four colors
can be measured; two colors for secondary beam excitation and one color
for tertiary laser excitation. This provides exceptional flexibility for
combining antibody measurement with other fluorochrome combinations such
as DNA dyes or for other fluorochrome measurements.
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Third laser window: We have found that coaxial beams
are not practical unless emissions are of similar intensity. To eliminate
this problem, we have designed a third laser window so that excitation/
emission occur at different times. This allows for resolution of fluorescence
based on different excitation characteristics even if the emission spectra
are identical.
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Cross beam compensation: One of the major disadvantages
of two laser flow cytometry has been the assumption that this compensation
would not be necessary because the fluorochromes excitation spectra would
not overlap. Not so, for example, the tandem conjugates, PE-TR, PE-Cy5
or PE-Cy7, are excited by both the primary 488 nM beam (PE) and by the
secondary beam (TR or Cy5, Cy7). In order to solve this problem as well
as to provide for compensation flexibility for the additional parameters,
we have added compensation networks to provide all possible fluorescence
parameter combinations.
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High speed sorting: A major limitation of flow cytometry
has been the inability to sort very large numbers of cells; usually bulk
separations are performed using magnetic beads, panning techniques or specific
binding columns for positive or negative selection. This may be followed
by cell sorting to improve purity, but often at an unacceptable cell loss.
Accordingly, we have developed a high speed cell sorter in collaboration
with BDIS currently marketed as the TurboSort Option.
There are two paradigms that need
to be clearly defined when we speak of high speed sorting. The first is
"high speed analysis" and slow speed sorting. This actually is the most
common form referred to as high speed sorting. In reality, however, this
is not high speed because the cells to be sorted are infrequent (often
less that 5%) and only conventional sorting speeds are required. For example,
if 1% of cells were desired, an analysis speed of 30,000 cells/sec. would
result in a sorting speed of only 300 cells/sec.
This paradigm is actually high speed cell analysis to increase throughput
but only conventional sorting speeds are required. This is very important
for stem cells and mixed populations in which the cell of interest represents
only a small fraction of the total. The second paradigm is truly high speed
sorting as the events to be sorted are a significant fraction of the total
(e.g. 50%). In this situation both high speed analysis and sorting are
required because an analysis speed of 30,000 cells/sec. would require a
sorting speed of 15,000/sec. Clearly, an instrument with this capability
would be able to handle any desired frequency. The FACSVantage with TurboSort
Option was conceived to meet these design features. A sort rate of 20,000
cells/sec. (7.2 x 107 cells/hr) at 98% purity and 70% recovery
are routine.
FACStar:
This instrument is used for conventional sorting and analysis of up to
three colors. It does not have UV excitation capabilities.
RESEARCH
FACScan: It is now possible to perform simultaneous four color
data acquisition using a single laser excitation source. The four fluorochromes
used for antibody conjugation are fluorescein isothiocyanate (FITC), phycoerythrin
(PE), PE-Texas Red (PE-TR) and PE-Cy5 or perdinin chlorophyll protein (PerCP).
The FACScan has the capability of measuring only three of these colors.
In order to accomplish four color measurements two major modifications
were required. In the first, a fifth photomultiplier tube (PMT) and associated
electronics were added including the necessary compensation networks. This
was wholly designed and built in our electronics laboratory. In the second,
the fixed filtration holders were changed to interchangeable filter holders.
This provides us the flexibility of changing filters and dichroic mirrors.
This instrument has been in use since 1993.
During the design of the compensation
networks, we addressed the problem of high autofluorescence associated
with many cultured cell lines. This problem can be completely solved by
electronic autofluorescence reduction on a cell by cell basis (Cytometry
7:566-574, 1986, Communications In Clinical Cytometry, 22:250-255, 1995).
Since the Research Scan allows for changeable optical filtration, we can
use one PMT for optimal detection of the desired fluorochrome and a second
PMT that accepts the more broadly distributed longer wavelength autofluorescence
emission that is outside the fluorochrome’s emission spectrum. The longer
wavelength autofluorescence emission is subtracted from the shorter autofluorescence
emission thereby nullifying autofluorescence. When the fluorochrome is
present, its fluorescence is detected with optimal efficiency regardless
of the cell’s autofluorescence.
Luminex FACScan: This instrument has the modifications
provided by Luminex to perform multiplex immuno and molecular assays using
fluorescent microspheres. In addition, the instrument can also be used
for standard five parameter single laser applications.
FACSCalibur:
This two laser, 6 parameter, 4 color instrument provides both clinical
research and reference laboratory service. The four fluorochromes most
often combined are FITC, PE, PE-CY5 (or PerCP) and APC. The entire data
acquisition is automated for operator free performance.
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