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Flow Cytometry Facility
Instrumentation
Sorting/Analysis Instruments:
MoFlo
EPICS Elite
MoFlo High-speed Sorter, DakoCytomation (purchased 2001) – Room G60/64
●Must be run by facility operators
●Two lasers; Argon (488nM) 100mW, Diode (635nM) 25mW
●Capable of sorting at rates up to 25,000 cells/second
●Capable of sorting 4 subpopulations simultaneously
Parameters available (most signals may be defined as linear or log):
●Forward Scatter;
●Right angle- (side) scatter
●Fluorescent signals - 6 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 6 colors
●Time and calculated parameters
●Inter-Laser delay, used for spatial separation of laser beams
PC-based operating and data analysis system, running under Windows NT
●Post-acquisition analysis package with options including listmode playback, histogram subtraction and overlays
Data Storage: hard drive, CD.
Network: access to Wistar network
Data may be analyzed using third-party flow cytometry software, such as CellQuest, WinMDI, or FlowJo
LSRII, Becton-Dickinson (purchased 2006) - Room 267
●Dedicated to investigator-operated analysis
●Two lasers; Argon (488nM) 20mW, Red HeNe (635nM) 20mW
●High-speed analysis-only instrument
Parameters available (all parameters linear or log):
●Forward Scatter
●Right Angle (side) Scatter
●Fluorescent signals - 7 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 7 colors
Suggested colors for immunofluorescence:
FITC
PE
PE-TxRED (R613, ECD)
PerCP or PE-Cy5 (R670, CyChrome)
PE-Cy7
APC
APC-Cy7, Alexa700, Alexa750
Fluorescent Proteins which may be analyzed:
GFP, YFP, DsRED
●Full compensation matrix, both inter- and intra-laser
●Time parameter
PC-based operating and data analysis system running under Windows
●Software (FACSDiva)
●Data may be analyzed using third-party flow cytometry software, such as FlowJo
Data Storage: hard drive, CD
Network: access to Wistar network
Data may be analyzed using third-party flow cytometry software, such as CellQuest, WinMDI, or FlowJo
CyAn ADP, DakoCytomation (purchased 2002) - Room G60/64
●Capable of “user-friendly” investigator-operation
●Three solid-state lasers (488nM, 635nM, 405 nM)
●High-speed (up to 30,000 events per second) analysis-only instrument
Parameters available (signals may be defined as linear, log, or peak):
●Forward Scatter;
●Right angle- (side) scatter
●Fluorescent signals - 9 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 9 colors
Suggested colors for immunofluorescence:
FITC
PE
PE-TxRED (R613, ECD)
PerCP, PerCP-Cy5.5, PE-Cy5 (R670, CyChrome)
PE-Cy7
APC
APC-Cy7, Alexa750, Alexa750
Cascade Blue
Cascade Yellow
Fluorescent Proteins which may be analyzed:
CFP, GFP, YFP, DsRED, HC-RED, FRET with CFP/YFP
●Full compensation matrix, both inter- and intra-laser
●Time parameter
PC-based operating and data analysis system (Summit software), running under Windows
●Software (Summit) is available for free from the facility
●Data may be analyzed using third-party flow cytometry software, such as WinMDI or FlowJo
Data Storage: hard drive, CD
Network: access to Wistar network
Data may be analyzed using third-party flow cytometry software, such as CellQuest, WinMDI, or FlowJo
FACSCalibur, Becton-Dickinson (purchased 2004) - Room G60/64
●Dedicated to investigator-operated analysis
●Two lasers; Argon (488nM), Red Diode (635nM)
Parameters available (all parameters linear or log):
●Forward Scatter
●Right Angle (side) Scatter
●Fluorescence 1, 2, 3 from 488nM Argon
●Fluorescence 4 (usually APC) from 635nM Red Diode
●Time and calculated parameters
●Doublet discrimination module
Power PC Mac G4/Dual Processor 1.25GHz
●Dedicated post-acquisition analysis package with options including listmode playback, cell cycle analysis, histogram subtraction and overlays Used for: investigator operation, multicolor immunofluorescence, DNA/RNA analysis (inc. apoptosis)
Data Storage: hard drive, CD
Network: access to Wistar network
Data may be analyzed using third-party flow cytometry software, such as CellQuest, WinMDI, or FlowJo
EPICS XL-MCL, BeckmanCoulter (purchased 1994) - Room G60/64
●Capable of “user-friendly investigator-operation
●Single Argon laser (488nM @15mW)
●Analysis-only instrument
Parameters available (all signals may be defined as linear or log):
●Forward Scatter;
●Right angle- (side) scatter
●Fluorescent signals - 5 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 5 colors
●Time and calculated parameters (ratio, PRISM)
MCL autoloader carousel allows for walkaway operation
PC-based operating and data analysis system, running under MS-DOS, compatible with Coulter EPICS-XL
●Post-acquisition analysis package with options including listmode playback, cell cycle analysis, histogram subtraction and overlays
Data Storage: hard drive, CD
Network: access to Wistar network
Data may be analyzed using third-party flow cytometry software, such as CellQuest, WinMDI, or FlowJo
FACScan, Becton-Dickinson (purchased 1994) - Room G60/64
●Dedicated to investigator-operation
●Single Argon laser (488nM @15mW)
Parameters available (all parameters linear or log):
●Forward Scatter
●Right Angle (side) Scatter
●Fluorescence 1, 2, 3
●Time and calculated parameters
●Doublet discrimination module
Macintosh Quadra650-based data analysis system, compatible with Mac
●Dedicated post-acquisition analysis package with options including listmode playback, cell cycle analysis, histogram subtraction and overlays Used for: investigator operation, multicolor immunofluorescence, DNA/RNA analysis (inc. apoptosis)
Data Storage: hard drive
Network: access to Wistar network
Data may be analyzed using third-party flow cytometry software, such as CellQuest, WinMDI, or FlowJo
Other related equipment available in Room G60/64:
Fluorescent microscope
Additional PC and Mac workstations for post-acquisition analysis
FlowJo post-acquisition software
Refrigerator
There is also a collection of the following publications and resources:
●Current Protocols in Cytometry
●Cytometry The Journal of the International Society for Analytical Cytology (1988 – current)
●Various flow cytometry reference books, including Practical Flow Cytometry (Shapiro, both 2nd & 3rd editions), Handbook of Flow Cytometry Methods (Robinson), and more
●Catalogs of flow cytometry-related supplies, antibodies, fluorochromes, etc.
Flow cytometry available elsewhere in The Wistar Institute
EPICS Elite, BeckmanCoulter (purchased 1991). Located in BSL3 laboratory.
●Must be run by trained operator
●Two lasers; Argon (488nM) 40mW, HeNe 10mW
●Capable of sorting at rates up to 5000 cells/second
Parameters available (all signals may be defined as linear or log):
●Forward Scatter;
●Right angle- (side) scatter
●Fluorescent signals - 5 PhotoMultiplierTubes (PMTs), which may be used in various configurations
●Time and calculated parameters (ratio, PRISM)
●Gated Amplifier, used for spatial separation of laser beams
PC-based operating and data analysis system, running under MS-DOS, compatible with Coulter EPICS-XL
●Post-acquisition analysis package with options including listmode playback, cell cycle analysis, histogram subtraction and overlays
Data Storage: hard drive
Data may be analyzed using third-party flow cytometry software, such as CellQuest, WinMDI, or FlowJo |
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