Director: Robert H. Carter, MD
Department/Center
Association: Medicine, Division of
Clinical Immunology and Rheumatology/AMC
Established: 1987
The
goal of the Flow Cytometry Core Facility is to
enhance the power and productivity of the research of UAB investigators by
providing flow cytometry for cell analyses and
purification at a reasonable cost. To
accomplish this goal, the Core provides the state‑of‑the‑art
equipment, service, and expertise necessary for the application of flow cytometry and related technologies to cutting edge
research.
Facility Description
The
AMC Flow Cytometry Core Facility is located in the
Instruments. The Core is equipped
with a FACScan, a FACSCalibur,
a FACSVantageSE with digital option (DiVa) and a LSRII.
All these cytometers are manufactured and
serviced by Becton Dickinson. The FACScan has a single argon ion laser emitting at
488nm. The instrument can measure up to
five parameters simultaneously. It can
be operated unassisted by users who have had prior training from the Core
Manager.
The
FACSCalibur is equipped with a 488nm air-cooled
argon-ion laser and a red diode laser emitting at 635nm. The instrument is capable of analyzing six
parameters (four colors, volume, and granularity).
The
FACSCalibur also has an automatic loader system
featuring 40 tube carousels and Worklist Manager
Software.
The
FACSVantageSE with digital option (DiVa) is a high speed fluorescence-activated cell sorter
(FACS) capable of analyzing up to 12 parameters (10 colors, volume, and
granularity). The instrument is equipped
with a Coherent Enterprise laser for 488nm and UV (360nm) excitation
wavelengths and a SpectraPhysics helium-neon laser,
which emits at 632nm. This three laser line
setup along with a variety of optical filters allows for simultaneous detection
of up to 10 colors. The sorting
capabilities of the FACSVantageSE have recently been
enhanced by the addition of the DiVa. The DiVa is a
digital operating system. Voltage pulses
are sent straight to the digital board from the pre-amplifiers. This results in improved signal linearity and
reduced processing time. The analytical
capacity has been increased from 8 to 12 parameters. There are full compensation networks for all
channels and all lasers. The QuadraSort feature allows the operator to sort 4-ways. Analysis is performed using a PC based DiVa software program.
In addition, the FACSVantage with DiVa is equipped with an Automatic Cell Deposition Unit
(ACDU) that sorts predefined numbers of cells onto microscope slides, or a variety
of microtiter plates (4, 24, 96 well) using CloneCyt Plus software.
The
LSR II is a four laser (405nm violet, 488nm blue, 535nm
green, 633nm red), 20-parameter analytic cytometer. The violet is especially suited for qdot excitation. The
green provides optimal excitation for phycoerthrin
(PE) and PE-containing tandem fluorochromes (PE/Alexa 610, PE/Cy5, PE/Cy5.5, PE/Cy7). The red can excite allophycocyanin
(APC) and APC tandems (APC/Cy7) as well as Alexa 647
and Alexa 700 fluorochromes. This machine also uses DiVa
digital software and can do automated compensation.
Research Information
Fee-Based Services. The Core has
equipment (BD Sample Prep Assistant and BD Wash/Lyse
Assistant) that provide automated staining of whole blood samples. This option should be considered for
quantitative flow cytometry or for clinical samples
acquired over time.
All other samples are prepared by the investigator. Consultation with the FACS facility personnel
is encouraged. “Current Protocols in Cytometry” is available in the facility as a reference
guide for choosing appropriate protocols and developing assays.
Some of the assays that are performed in this facility are: immunophenotyping with the use of up to 3 (FACScan), 4 (FACSCalibur), or 18
(LSRII) fluorochromes. On the FACSVantageSE
with DiVa option there is the potential to perform
analyses of up to 10 colors. Services
include intracellular immunofluorescence, including
intracellular cytokine analysis; cell cycle analysis; apoptosis assays;
analysis of GFP expression; viability assays; sterile cell sorting; single cell
cloning; calcium flux assays; mitochondrial function assays; quantitation of cytokines; and oxidative metabolism assays.
The
fees for analysis are as follows:
·
FACScan: $50/hour, $35/hour
if analyzed by the tutored investigator
·
FACSCalibur: $75/hour, $35/hour
self run
·
FACSVantageSE with DiVa option: $75/hour
·
LSRII: $60/hour, $50/hour self run
·
Tutorial on
the FACScan (usually requires two 1-hour
sessions): $50/hour
Scheduling. FACSCan
time may be scheduled online at http://tesla.cdfi.uab.edu/flow1.
Investigators must submit account numbers so that passwords may be
assigned. Appointments for the other
machines should be made at least 24 hours in advance, as should notice of
cancellations. If investigators plan a
large experiment (over 60 tubes) they must notify the facility a week in
advance. Appointments for sterile sorts
should also be made at least a week in advance.
The AMC Flow Cytometry Core Facility is supported by NIH P30 AR48311.
Core Director: Robert
H. Carter, MD
Email: Robert.Carter@ccc.uab.edu
Phone: 205-975-8688
Core Manager: Enid F.
Keyser, MS
Email: enid.keyser@ccc.uab.edu
Phone: 205-934-1362
Website: http://www.uab.edu/laserlab
Approved by: Robert
H. Carter, MD, Director
Date: April 15, 2008
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