FLOW CYTOMETRY FACILITY
Director:
Jianke Zhang, PhD
Tel: (215) 503-4559
|
Location:
606 BLSB
233 S. 10th Street
Philadelphia, PA 19107
|
Contact:
Matthew Farabaugh
Tel: (215) 503-4556
|
The Kimmel Cancer Center Flow Cytometry facility provides state-of-the-art cell sorting and analysis capabilities to investigators of the Kimmel Cancer Center and the Philadelphia region.
The most commonly used applications are multi-color cell surface phenotyping, detection of intracellular cytokines and signaling molecules, transfection measurements, studies of apoptosis, cell cycle analysis, cell proliferation, and real time analysis of Ca2+ mobilization kinetics.
The sorters, analyzers, and workstations allow for affordable, reliable, timely, and accurate characterization of a wide variety of biological samples.
The services provided by the facility can be tailored to suit experimental designs in a variety of fields. We provide consultation about current protocols, analysis techniques, and data presentation.
This resource plays a critical role in assisting KCC members performing various research projects directly related to the understanding of the biology and treatment of cancer. Furthermore the facility supports projects from investigators in Biochemistry, Genetics, Immunology, Medicine, Microbiology, Molecular Biology, Neurology, Pathology, Pharmacology, Surgery, and many other scientific fields.
Multiple publications have been accepted in journals with the data and results generated by the analyzers and sorters in the facility.
Typical applications include multiparamter analysis such as the flow data shown on the right.
Cells were analyzed using the Coulter EPICS XL-MCL and the figure was generated with FlowJo software.
Results show B cell proliferation during an immune response using CFSE labelling.
B, Rahman ZS, and Manser T. J Immunol. 2007 May 1;178(9):5623-34
A variety of kinetics assays can also be performed such as the calcium influx experiment shown here.
Cells were analyzed using the BD FACSCalibur and the figure was generated with FlowJo software.
Results show calcium influx on CD16 triggered NK cells with and without Prostaglandin treatment.
Chen Y, Perussia B, Campbell KS. J Immunol. 2007 Sep 1;179(5):2766-73
The data on the right shows a typical two fluorochrome immuno-stain performed on the Coulter Epics XL-MCL.
Mouse bone marrow cells and splenocytes were analyzed for levels of Gr-1myeloid differentiation markers and transcription factors for granulocyte differentiation.
The GFP positive cells were sorted on the Dako MoFlo sorter and a subsequent cell cycle anaylsis was performed by propidium iodide staining.
Both the proliferation and differentiation capabilities of these mouse splenocytes and hematopoietic cells were assessed.
Ferrari-Amorotti G, Keeshan K, Zattoni M, Guerzoni C, Iotti G, Cattelani S, Donato NJ, Calabretta B. Blood. 2006 Aug 15;108(4):1353-62
Flow Cytometry is a continually expanding field and listed below is a brief table of biological events that can be measured in the facility.
| PARAMETER |
MEASUREMENT METHOD |
| Intrinsic Parameters |
(no probes needed) |
| Cell Size |
Forward Light Scatter |
| Cytoplasmic Granularity |
Right Angle (or side) Light Scatter |
| Cell Shape and Aggregation |
Pulse Shape Analysis |
| Autofluorescence |
Fluorescence |
| Autofluorescence |
Fluorescence |
| |
| Extrinsic Parameters |
(probes needed) |
| Antigens |
Fluorescence (labeled antibodies) |
| DNA Content |
Fluorescence (e.g. PI) |
| Nucleic Acid Sequence |
Fluorescence (e.g. EGFP, β-gal) |
| RNA Content |
Fluorescence (e.g. AO) |
| Surface Sugars |
Fluorescence (labeled lectins) |
| Membrane Integrity (viability) |
Fluorescence (PI) |
| DNA Degrradation (apoptosis) |
Fluorescence (e.g. PI) |
| DNA Synthesis |
Fluorescence (anti-BrUdR antibodies) |
| Cytokines |
Fluorescence (labeld antibodies) |
| Cell Division |
Fluorescence (CFSE) |
| Conjugate Formation |
Fluorescence (HE, calcein, etc.) |
| Surface Receptors |
Fluorescence (labeled ligands) |
| Membrane Organization |
Fluorescence (Annexin) |
| Intracellular Receptors |
Fluorescence (labeled ligands) |
| Cytoplamic Ca++ |
Fluorescence (indo-1) |
| Intracellular pH |
Fluorescence (e.g. SNARF) |
INSTRUMENTS
| Type: |
Dako Cytomation MoFlo |
| |
High Performance Cell Sorter |
| Software: |
Summit |
| Features: |
2 lasers, High Speed |
| |
Sorting, 7 Colors |
| Lasers: |
488nm air-cooled Argon-ion |
| |
633nm red diode |
| Detectors: |
530nm(FITC/GFP/Alexa488) |
| |
585nm(PE) |
| |
630nm(PE-Texas Red) |
| |
670nm(PerCP/PerCP5.5/TC) |
| |
740nm(PE-Cy7) |
| |
670nm(APC) |
| |
740nm(APC-Cy7) |
| Sorting: |
> 95% Purity, 25,000 cells/sec |
| Type: |
BD FACSCalibur |
| Software: |
CellQuest |
| Features: |
2 Lasers, Low Speed Sorting |
| |
4 Colors |
| Lasers: |
488nm air-cooled Argon-ion |
| |
633nm red diode |
| Detectors: |
530nm(FITC/GFP/Alexa488) |
| |
585nm(PE/PI) |
| |
650nm(PerCP/PECy5) |
| |
670nm(APC) |
| Sorting: |
> 95% Purity, 300 cells/sec |
| Type: |
Coulter Epics XL-MCL |
| Software: |
XL System II |
| Features: |
1 Laser, Multi-sample Carousel Loader |
| |
4 Colors |
| Laser: |
488nm air-cooled Argon-ion |
| Detectors: |
530nm(FITC/GFP) |
| |
585nm(PE/TC) |
| |
620nm(PI) |
| |
670nm(PerCP/PECy-5) |
| Type: |
Coulter Epics Elite ESP |
| Software: |
Expo 32 |
| Features: |
2 Lasers, Low Speed Sorting |
| |
7 Colors, BSL-3 |
| Lasers: |
488nm air-cooled Argon-ion |
| |
633nm Helium-Neon Laser |
| Detectors: |
530nm(FITC/GFP/Alexa488) |
| |
585nm(PE) |
| |
630nm(PE-Texas Red) |
| |
670nm(PerCP/PerCP5.5/TC) |
| |
740nm(PE-Cy7) |
| |
670nm(APC) |
| Sorting: |
> 90% Purity, 5,000 cells/sec |
Analysis Software
Available Antibodies and Dyes
Background and Protocols
Fluorochrome Compatability
Fluorochrome Tables
