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Flow Cytometry Facility
Instrumentation
Sorting/Analysis Instruments:
FACSAria II
MoFlo
SORTING INSTRUMENTS
FACSAria II cell sorter, BD (purchased 2009) – Room G60/64
- Operated by facility personnel only
- Three lasers; Blue (488nM) 20mW, Red (633nM) 20mW, UV (375nM) 50mW
- Capable of sorting at rates up to 21,000 cells/second (70u) or 9,000 cells/sec (100u)
- Capable of sorting up to 4 subpopulations simultaneously
Parameters available (most signals may be defined as linear or log):
- Forward Scatter
- Side (right angle)- scatter
- Fluorescent signals - 11 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 11 colors simultaneously, with full compensation matrix
- Time and calculated parameters
Suggested fluorochromes:
(Blue Laser) FITC/GFP
PE
PE-TxRed or PI
PerCP-Cy5.5/PE-CY5.5/ PE-CY5/ PerCP/7AAD
PE-Cy7
(Red Laser) APC
ALEXA 700
APC-Cy7/ ALEXA 750
(Ultra Violet) Hoechst Red
Hoechst Blue/ DAPI
Contact facility for other fluorochromes not listed, or with questions regarding color combinations
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 and Wistar network servers
MoFlo High-speed Sorter, DakoCytomation (purchased 2001) – Room G60/64
- Operated by facility personnel only
- Two lasers; Argon (488nM) 100mW, Diode (635nM) 25mW
- Capable of sorting at rates up to 25,000 cells/second (70u) or 10,000 cells/sec (100u)
- Capable of sorting up to 4 subpopulations simultaneously
Parameters available (most signals may be defined as linear or log):
- Forward Scatter;
- Side (right angle)- scatter
- Fluorescent signals - 6 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 6 colors simultaneously with full compensation matrix
- Time and calculated parameters
Suggested fluorochromes:
(Blue Laser) FITC/GFP
PE
PE-TxRed
PerCP-Cy5.5/PE-CY5.5/ PE-CY5/
PerCP/7AAD
(Red Laser) APC
ALEXA 700 or APC-Cy7/ ALEXA 750
Contact facility for other fluorochromes not listed, or with questions regarding color combinations
PC-based operating and data analysis system running under Windows
- Software (Summit)
- Data may be analyzed using third-party flow cytometry software, such as FlowJo
Data Storage: hard drive and Wistar network servers
ANALYSIS INSTRUMENTS
LSRII (14-color), Becton-Dickinson (purchased 2006) - Room 26
- Dedicated to investigator-operated analysis
- Four lasers; Argon (488nM) 20mW, Red HeNe (635nM) 20mW, Green (532mM), Violet (405nM)
- High-speed analysis-only instrument
Parameters available (all parameters linear or log):
- Forward Scatter
- Side (right angle)- scatter
- Fluorescent signals - 14 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 14 colors simultaneously with full compensation matrix
- Time and calculated parameters
Suggested fluorochromes:
(Blue Laser) PerCP 5.5
PE-BIue
FITC
(Green Laser) PE-CY7
PE-CY5.5
PE-CY5
PE-TxRed
PE-Green
(Red Laser) APC-Cy7
ALEXA 700
APC
(Violet) PacBlue
AmCyan
Qdot605 or Qdot655
Fluorescent Proteins which may be analyzed:
GFP, YFP, CFP, DsRED, other monomeric rfp’s
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 and Wistar network servers
LSRII (18-color), Becton-Dickinson (purchased 2006) - Room 267
- Dedicated to investigator-operated analysis
- Four lasers; Argon (488nM) 20mW, Red HeNe (635nM) 20mW, Green (532mM), Violet (405nM)
- High-speed analysis-only instrument
Parameters available (all parameters linear or log):
- Forward Scatter
- Side (right angle)- scatter
- Fluorescent signals - 18 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 18 colors simultaneously with full compensation matrix
- Time and calculated parameters
Suggested fluorochromes:
(Blue Laser) PerCP 5.5
FITC
(Green Laser) PE-CY7
PE-CY5.5
PE-CY5
PE-TxRed
PE-Gr
(Red Laser) APC-Cy7
ALEXA 700
APC
(Violet) Qdot 800
Qdot 705
Qdot 655
Qdot 605/625
Qdot 585
Qdot 565
AmCyan
PacBlue
Fluorescent Proteins which may be analyzed:
GFP, YFP, CFP, DsRED, other monomeric rfp’s
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 and Wistar network servers
CyAn ADP, DakoCytomation (purchased 2002) - Room G60/64
- Capable of investigator-operated analysis
- Three solid-state lasers (488nM, 635nM, 405nM)
- 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;
- Side (right angle)- scatter
- Fluorescent signals - 9 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 9 colors simultaneously with full compensation matrix
- Time and calculated parameters
Suggested fluorochromes:
FITC
PE
PE-TxRED (R613, ECD)
PerCP, PerCP-Cy5.5, PE-Cy5
PE-Cy7
APC
APC-Cy7, Alexa750, Alexa750
Cascade Blue, PacBlue
Cascade Yellow
Fluorescent Proteins which may be analyzed:
GFP, YFP, CFP, DsRED, other monomeric rfp’s
Contact facility for other fluorochromes not listed, or with questions regarding color combinations
PC-based operating and data analysis system running under Windows
- Software (Summit)
- Data may be analyzed using third-party flow cytometry software, such as FlowJo
Data Storage: hard drive and Wistar network servers
FACSCalibur, Becton-Dickinson (purchased 2004) - Room G60/64
- Dedicated to investigator-operated analysis
- Two lasers; Argon (488nM), Red Diode (635nM)
- Low-speed (up to 5,000 events per second) analysis-only instrument
Parameters available (all parameters linear or log):
- Forward Scatter
- Side (right angle)- scatter
- Fluorescence 1, 2, 3 from 488nM Argon
- Fluorescence 4 (usually APC) from 635nM Red Diode
- Time and calculated parameters
- Doublet discrimination module
Suggested fluorochromes:
FITC
PE
PerCP, PerCP-Cy5.5, PE-Cy5/PI
APC
Fluorescent Proteins which may be analyzed:
GFP, YFP, DsRED, other monomeric rfp’s
Mac- based operating and data analysis system
●Software (CellQuest)
●Data may be analyzed using third-party flow cytometry software, such as FlowJo
Data Storage: hard drive and Wistar network servers
EPICS XL-MCL, BeckmanCoulter (purchased 1994) - Room G60/64
- Capable of investigator-operated analysis
- Single Argon laser (488nM @15mW)
- Low-speed (up to 5,000 events per second) analysis-only instrument
- MCL autoloader carousel allows for walk-away operation
Parameters available (all signals may be defined as linear or log):
- Forward Scatter;
- Side (right angle)- scatter
- Fluorescent signals - 5 PhotoMultiplierTubes (PMTs), which may be used in various configurations, capable of up to 5 colors
- Time and calculated parameters
Suggested fluorochromes:
FITC
PE
PE-TxRed/PI
PerCP, PerCP-Cy5.5, PE-Cy5
Fluorescent Proteins which may be analyzed:
GFP, YFP, DsRED, other monomeric rfp’s
PC-based operating and data analysis system, running under MS-DOS compatible with Coulter EPICS-XL
- Software (XL SystemII)
- Data may be analyzed using third-party flow cytometry software, such as FlowJo
Data Storage: hard drive and Wistar network servers
FACScan, Becton-Dickinson (purchased 1994) - Room G60/64
●Dedicated to investigator-operated analysis
●Single Argon laser (488nM @15mW)
●Low-speed (up to 5,000 events per second) analysis-only instrument
Parameters available (all parameters linear or log):
●Forward Scatter
●Side (right angle)- scatter
●Fluorescence 1, 2, 3 from 488nM Argon
●Time and calculated parameters
●Doublet discrimination module
Suggested fluorochromes:
FITC
PE
PerCP, PerCP-Cy5.5, PE-Cy5/PI
Fluorescent Proteins which may be analyzed:
GFP, YFP, DsRED, other monomeric rfp’s
Mac- based operating and data analysis system
- Software (CellQuest)
- Data may be analyzed using third-party flow cytometry software, such as FlowJo
Data Storage: hard drive and Wistar network servers
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.
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