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Flow Cytometry/Cell Sorter
Welcome to the RFUMS Flow Cytometry Core Facility, operated by the Center for Cancer Biology, Immunology, and Infection.
Hours of operation are 8:00a.m.- 5:00p.m. M-F.
Special hours of operation requests MUST be made no less than SEVEN business days before requested sort, and APPROVED before an individual should start any long-term experiments.
Prior to running samples on any instruments all projects require approval by RFUMS-EHS.
Flow cytometry is a technology capable of analyzing multiple characteristics of individual cells at a rate of thousands of cells per second. This is accomplished by transporting the individual cells through an illuminating light source, usually a laser, by a fluid stream and then electronically collecting the resulting signals. In conjunction with fluorescently tagged monoclonal antibodies and physiological dyes, clinical laboratories use cytometers to detect lymphomas, leukemia, DNA ploidy, and immunodeficiency disorders. In research they are used for the detection of protein expression, apoptosis, cell cycle analysis, calcium ion flux, reactive oxygen species and numerous other applications. Flow Cytometers equipped for cell sorting can physically separate one cell from another based on cellular characteristics. This technology can be used to sort stem cells, transfected cell lines, or a specific cell population for later experimentation. The core flow cytometry and cell sorting laboratory here at RFUMS is equipped with state-of-the-art flow cytometers for both analysis and sorting. These cytometers are described below.
All new RFU users are required to go through 3 training sessions to learn to use the analyzers (not the cell sorter). The first session will be an overview of the analyzer, SOP for the core, and more; no samples during the first session. The second and third sessions are for the user to bring their own samples and learn how to set up the software for acquisition, set up, and run the analyzer, and export data to use in FlowJo for analysis. After completing these trainings, the user will be evaluated if they can use the analyzers unassisted going forward or require more training.
For more detailed information concerning flow cytometry please see the links listed below.
Introduction to Flow Cytometry Sites
Tool for planning multi-color experiments
Cytek Aurora CS |
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The Cytek Aurora CS (CS) is a state-of-the-art spectral cell sorter that can collect from 2 to 6 different specific populations of cells from a mixed sample into various sized collection tubes (15ml conical= max 2-way sort; 5ml round bottom tube= max 4-way sort; 1.5ml flip cap tubes= max 6-way sort). It is also possible to sort into 96 well plates. The CS is equipped with 4 lasers (405nm violet, 488nm blue, 561nm yellow-green, 640nm red) and is capable of detecting 48 different fluorescent channels plus FSC, blue laser SSC, and violet laser SSC. CS utilizes the latest version of Spectroflo® CS sorting software. The collection chamber has integrated HEPA filtration to reduce the risk of aerosols. The entire unit is also contained within a bioBubble Class 1 Benchtop Biocontainment Enclosure (see below). All sorts require prior consultation with either the flow core technician or directors of the flow core, and scheduling with the flow core technician at least 1 week before the scheduled sort.
Cancellation of any scheduled sorts must be given at least 24 hours in advance, or the individual will be charged for half the scheduled time.
bioBubble Class I Benchtop Biocontainment Enclosure (BBE)
The CS is contained within the bioBubble BBE to protect the operator and laboratory personnel from possible exposure to aerosols created by the sorter. Metal scaffolding is assembled around the sorter and from this scaffolding the containment plastic, fans and HEPA filters are suspended. The bioBubble uses these fans to draw air from the laboratory into the BBE and out through HEPA filters. The bioBubble also evacuates aerosols directly from the flow chamber. The containment of the CS in the BBE allows for sorting primary human samples and other materials that are handled using BSL2 precautions.
Cytek Aurora |
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The Cytek Aurora (Aurora) is a 4 laser (405nm violet, 488nm blue, 561nm yellow-green, 640nm red) benchtop spectral analytical cytometer to support large applications, with 48 color channels plus FSC, blue SSC, and violet SSC. This cytometer utilizes Spectroflo® software for acquisition and analysis. Files can be exported as .fcs 3.1 type files and are compatible with FlowJo.
LSR II |
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LSRII is a three laser (488nm Coherent sapphire blue, 635nm HeNe and 405nm Coherent violet laser) conventional analytical benchtop system designed to support basic through advanced flow cytometric applications in up to 10 colors (12 parameters currently). The ability to reconfigure the optics in this machine provides extra flexibility in experimental design. This cytometer utilizes PC based FACS Diva acquisition and analysis software, although acquired data can be analyzed in CellQuest Pro also. ModFit Program, for DNA analysis is also available.
autoMACS Pro Separator |
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Anyone interested in using an AutoMACS Pro should contact Dr. Joseph Reynolds (joseph.reynolds@rosalindfranklin.edu).
Flow Data Analysis Stations |
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For new users and infrequent users, there are two standalone computers available for data analysis. We have a Mac with Flowjo software, and a PC with Spectroflo software. These are available for all RFU users.
Verity’s Modfit is for analyzing cell cycle, cell proliferation and cell synchronization data. Its data models can automatically analyze your data, or you can manually adjust models to analyze it. The automatic models correct for debris, aggregates and non-linearity. For further detailed explanations and tutorials on this program, see the link below.
Flowjo analysis program can group sample data by experiment and then apply gates from one sample to all samples within the group. Flowjo’s workspace window allows you to view all statistics and gates of a group in a hierarchical view. Data can be displayed as histogram or dot plot overlays where more than one sample is presented in the same display. This allows for direct comparison of fluorescent intensity. For further detailed explanations and tutorials on this program, see the link below.
Applications of Flow Cytometry
- Detection of surface markers
- Detection of intracellular markers (cytoplasmic and nuclear)
- DNA cell analysis
- Cell proliferation
- Intracellular cytokine analysis
- Apoptosis assays
- Cell sorting - transfectants and other population
| User Fees* | ||
|---|---|---|
| RFUMS Users | Non-RFUMS Users | |
| LSRII | Assisted - $60/hr. Unassisted - $40/hr. |
Assisted - $120/hr. Unassisted - $80/hr. |
| Sorting (CS) | Set-up - $77/hr. Assisted - $77/hr. Unassisted - $62/hr. |
Set-up - $155/hr. Assisted - $155/hr. Unassisted - $124/hr. |
| Cytek Aurora | Assisted - $55/hr. Unassisted - $33/hr. |
Assisted - $100/hr. Unassisted - $55/hr. |
| Consulting: Panel design, Data analysis, extra training, etc. |
$25/hr. | $100/hr. |
| *Based on 15-minute intervals. All unassigned users must be trained and/or authorized by facility staff. Training is available. | ||
Scheduling
The RFU Flow core utilizes Google Calendars for all scheduling of its instruments. Upon completion of training on the analyzers, users will be granted access to the analyzer calendars to schedule their experiments.
CS scheduling is based on approval that the time works for the flow core employees and is not already booked for another user. RFU users will be granted access to see CS events, but not add or edit events.