Instrumentation:
Upgraded 5-Color FACScan Analyzers (2 available):
- A bench top analyzer with two lasers for
fluorochrome excitation: The primary laser is a 15 mw argon (488 nm) laser
and the secondary laser is a 25 mW red diode (637 nm) laser.
- Seven detectors: Two for light scatter (forward and
90°) and five for fluorescence. The optical
filters for the fluorescence detectors are fixed at FL1 = 530±15 nm, FL2 =
585±21 nm, FL3 = 668 nm long pass, FL4 = 666±13 nm and FL5 = 740 nm long
pass.
- Suitable for immunofluorescence (up to 5 color) and
DNA content/cell cycle analysis. A link to a pdf covering basics of
instrument operation is available below.
- Commonly used fluorochromes (please note that
Texas Red can not be used as a
single fluorochrome on these instruments):
Download a
"Flurochrome Guide" pdf
Download "FACScan Operation
Guidlines" pdf
FACSVantage SE High Speed Cell Sorter:
- A dual laser instrument (argon and helium-neon
lasers) capable of generating three simultaneous laser beams for
fluorochrome excitation (UV at 340-360nm, blue at 488nm and red at 633nm).
Please note that Texas Red can not be used
as a fluorochrome with these lasers! Also,
please be aware that PerCP is a very weak fluorochrome
on the Vantage SE cell sorter and should not be used unless you are
working with a very brightly staining marker. Instead of PerCP you can
use the tandem dye
PerCP-Cy5.5 which was specifically designed as an FL3 fluorochrome
for jet-in-air cell sorters.
- Ten detectors: Two for light scatter (forward and 90°)
and eight for fluorescence. The fluorescence detectors are arranged so
that four receive signals from the primary 488 nm beam, two from the
second 633 nm beam and two from the third 340-360 nm beam. Optical filters
may be changed on each of these fluorescence detectors to capture a
particular fluorescence emission wavelength.
A maximum of six fluorescent signals can be collected at one time.
- Cell sorting capabilities for two populations defined
by any combination of light scatter and fluorescence characteristics. The
maximum cell analysis rate for sorting is 8 x 106 cells/hour. To
view more information about cell sorting
click here.
- A recirculation water bath is available to maintain
the sample tube and the sorted cells at a defined temperature.
- An Automatic Cell Deposition Unit (ACDU) option
allows the sorting of defined numbers of cells into the wells of a
microtiter or other tissue culture plate.
Data Analysis:
Computers:
Each cytometer is operated by a Macintosh G4
computer equipped with a Fujitsu 235 Mb magneto-optical disk drive and 100 Mb
ZIP disk drive for data transfer and storage. Those bringing disks for data
storage should be sure to format them for Macintosh use. Data
analysis service is available by core lab personnel or investigators may use one
of our off-line workstations to perform their own data analysis. Off-line
workstations are located at:
- FACScan Lab in room 603 Wintrobe (available on
first-come first-served basis).
- Department of Pathology in room 5B124 SOM.
- Huntsman Cancer Institute, Samlowski Lab area in room
5254 (ask for John McGregor for help locating the computer).
- Eccles Institute for Human Genetics (EIHG), Zimmerman
lab in room 4250A.
Software:
Software available includes Becton-Dickinson's
CellQuest, the basic data acquisition and analysis software, on all
off-line workstations. In addition, available on the workstation in 603
Wintrobe is a DNA/Cell Cycle modeling program ModFit, a more
sophisticated flow cytometry data analysis program WinList, and an
antigen quantitation program QuantiCalc all three from Verity
Software House.
Support Services:
Full service by lab personnel is available for
staining, analyzing and data analysis during normal working hours. Training is
provided for individuals who wish to operate the FACScans themselves and take
advantage of off-hour access. Collaborative support is also available for
developing new assays and applications of the technology.