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Flow Cytometry Core 
FACSAria The Flow Cytometry Core is one of the many core facilities at the
Sidney Kimmel Cancer Center. Our purpose is to provide technical assistance
to SKCC investigators and the surrounding research community for performing
flow cytometry based analysis and sorting. With state of the art instrumentation,
such as the FACSAriaTM, the facility offers high speed sorting and
complex multi-color analytical services. In addition, the FACSCaliburTM can provide up to four color routine analyses. Investigators have the
option, once trained, of performing their own analyses using the FACSCaliburTM or utilizing the experstise of the core facility’s staff to run
their samples for them. The FACSAria, however, is run solely by the
designated operator. The applications of flow cytometry are numerous,
and this has led to the widespread use of the technology in scientific
and medical fields. More about flow cytometry and the core facility
can be found by clicking on the appropriate links. Personnel: Phyllis-Jean Linton, Director
Carina Rumold, Manager/Operator Table of Contents:
I. What is Flow Cytometry?
II. What is Flow Sorting?
III. The Facility
A. Instruments
1. FACSCaliburTM
2. FACSAriaTM
B. Data Analysis
C. Training
D. Contact Us What is Flow Cytometry?
Flow cytometry uses fluorescent probes, which bind to specific cell
associated molecules, to measure multiple physical characteristics
of individual cells (or particles) in suspension. As labeled cells
flow past a laser beam, the probes fluoresce and the emitted light
is directed to detectors which translate the light signals into information
concerning the amount of probe associated with each cell. Information
regarding the relative size and granularity of a cell as well as
any cell component or function that is detectable via a fluorescent
compound can be examined.
What is Flow Sorting?
Fluorescence Activated
Cell Sorting (FACS) is a powerful tool used to purify cell sub-populations
based on properties
measured and defined during flow analysis. The ability to electronically
deflect cells with preset, defined properties into a separate collection
tube provides the basis for flow sorting. Once cell populations are
identified, cells can be sorted at very high purity (>95%). The
ability to detect up to 13 fluorochromes simultaneously allows complex
mixtures of cells to be separated based on expression of multiple markers.
The SKCC Flow Cytometry Core Facility
The Flow Cytometry Core Facility is located on the second floor of
Building #1 in approximately 300 square feet of space. The facility
is equipped with an analytical instrument (FACSCalibur) and two MAC
computer workstations. Recently, a preparative cell sorter, i.e.,
FACSAria (sorter), and a PC computer workstation have been added
to the facility.
FACSCalibur FACSCaliburTM
The FACSCalibur, purchased from an NIH equipment grant
in l996, is a highly sensitive dual laser, nonsorting instrument used
exclusively for quantitative analysis applications. The instrument
is equipped with one laser that excites at 488nm and a second diode
laser that excites at 633nm. Because the FACSCalibur is equipped with
four fluorescence detectors, simultaneous analysis of six parameters
(two light scatter and four fluorochromes) is routine. Four fluorochromes
can be combined, three of which excite at 488nm and the fourth at 633nm.
The electronics allow for inter-laser compensation between FL3 and
FL4, thus, enabling experiments using fluorochrome combinations such
as FITC/PE/Red613/APC, FITC/PE/PerCP/CY5, FITC/PE/Cy5-PE/APC, and FITC/PE/PI/APC.
The FACSCalibur is engineered with fixed optical, electronic and fluidic
components, giving it the flexibility to function as an investigator-operated
instrument.
FACSAriaTM
The BD FACSAria is a benchtop sorter that incorporates
a fixed-alignment cuvette flow cell. This new flow cell provides superior
fluorescence sensitivity. The fixed optical system offers freedom from
instrument maintenance and alignment. The FACSAria has up to three
air-cooled lasers at 488nm, 633nm and 407nm allowing multicolor analysis
of up to15 parameters. Sort setup and sort monitoring features in the
system software make the BD FACSAria instrument easier to use than
the FACSVantage SE II. BDTM AccuDrop is integrated into the system
for quick and accurate drop-delay determination. Stream monitoring
of the breakoff point and clog detection are incorporated for walkaway
sorting. The instrument has digital acquisition rates of up to 70,000
events/second. Sort samples can be collected in two- and four-way bulk
sorting devices with a variety of tube sizes or with the BD Automated
Cell Deposition Unit (ACDU) for sorting to multiwell plates or microscope
slides. Because the FACSAria does not have a UV laser, calcium flux
assays with Indo dyes cannot be done.
The flow facility staff is responsible for maintaining the instruments
in optimal working order and training investigators in their use. The
analytical instruments can be operated by the users or facility staff,
should the user opt, while the FACSAria sorter will be operated solely
by the trained flow facility staff members. Sign up sheets are posted
for reserving time on the analytical instruments on a first come first
served basis. The instruments have a File Guard Security System. Accounts
are obtained from the facility staff. To reserve time on the preparative
cell sorter, investigators should contact the flow facility staff. Data Analysis
Individual investigators are responsible for their own data analysis.
Within the flow facility are three workstations and software options
for users. The user operated flow cytometer (FACSCalibur) has Macintosh
based computer systems. The two Macintosh G4 workstations have Cell
Quest, FloJo and ModFit LT software for data analysis. The FACSAria
cell sorter is run by a PC based computer system. There is one PC
workstation with Diva software for data analysis and/or for setting
up sort profiles. The computers for data acquisition are networked
to a NT Windows server having a 24-gigabyte storage capacity. Investigators
are encouraged to store their data on the server so that it can be
accessed from laboratory computers. Alternatively, data may be saved
on a zip disk or CD-ROM. There is no charge for the use of the workstations
and both color and black and white laser printers, located throughout
the Center, are available through a printer server network. The facility
staff is available when needed to assist in data analysis.
Training
Providing training and education for all flow users is a primary
goal of the facility staff. Due to the highly technical nature of
flow
cytometry, mistakes made during acquisition and/or data analysis
are common among untrained users. Group training sessions (at several
levels) are available and individual help is provided whenever possible.
Contact staff personnel for specific information regarding training
sessions. Typically, new SKCC staff that will use the flow cytometry
facility are given an orientation to cover the broad scope of facility
use and some specifics on the use of the cytometers as well as individualized
training using the researcher’s samples (~1-2 hour training
session). Users that want additional training or people new to flow
cytometry are encouraged to contact the director for personal assistance
during several data acquisition and analysis sessions before working
extensively on their own.
For further information or to schedule
time, contact Carina Rumold 858-450-5990 extension 233 or crumold@skcc.org
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