Flow Cytometry /Cell sorter
Welcome to the RFUMS Flow Cytometry Core Facility
The Flow Cytometry Core Facility is operated by the Department of Microbiology and Immunology.

 

Prior to running samples on the 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 flow cytometers for both analysis and sorting. These cytometers are described below.

For more detailed information concerning flow cytometry please see the links listed below.

Introduction to Flow Cytometry Sites

BD Biosciences

Beckman Coulter

Life Technologies

Tool for planning multi-color experiments

BD Biosciences

FACS Aria II Cell Sorter

The FACS Aria II is a benchtop high speed cell sorter that can acquire up to 70,000 events/sec. It incorporates a fixed alignment cuvette flow cell with a fixed optical system and is armed with three lasers: (488nm argon blue , 633nm HeNe red,  and 405nm violet ). This permits multicolor detection of up to 9 colors along with FSC and SSC. The instrument can sort into 2 or 4 tubes, of various sizes, as well as into multiwell plates or slides. The FACSAria II utilizes the latest version of PC-based FACSDiva software, which provides for enhanced sorting control and flexibility and data analysis. All sorts require a prior consultation with the laboratory manager and scheduling with the laboratory manager at least 48 hours before the sort.

LSRII 

LSRII is a three laser (488nm Coherent sapphire blue, 635nm HeNe and 405nm Coherent violet laser) 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.

Gallios

The Gallios flow cytometer is a high speed 3 laser (Violet 405nm, Argon Blue 488nm, and HeNe Red 638nm) 10 color research instrument which supports beginning to advanced flow cytometry applications The Gallios has a wide or narrow angle mode for the acquisition of forward scatter data.  Because of this, the Gallios can detect particles in the size range of.400μm to 40μm.  Acquired data can be analyzed in Kaluza software as well Modfit and FlowJo.   The link below gives the standard filters and fluorochromes used with the Gallios.

Instrument Fluorochrome Guide

All three of our flow cytometers store acquired data as flow cytometry standard data 3.0 list mode files (FCS 3.0).  These standard format files can be analyzed by all three of the independent software packages we have.  There is no charge for using the analysis software.

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.

Verity Software House

TreeStar’s Flowjo analysis program can group sample data by experiment and then apply gates from one sample to all samples within the group.  Flowjo’s work space 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.

Flowjo Tutorials

Beckman Coulter’s Kaluza software can analyze large files quickly and efficiently. It can display multiple samples within a work space and overlay histograms to display multiple samples within the same view.  It can easily copy plots from one experiment to another for easy comparisons. For further detailed explanations and tutorials on this program, see the link below.

Kaluza Software

APPLICATIONS OF FLOW CYTOMETRY

- Detection of surface markers
- Detection of intracellular markers (cytoplasmic and nuclear)
- DNA cell analysis
- Intracellular cytokine analysis
- Apoptosis assays
- Cell sorting - transfectants and other population

USER'S FEES*

RFUMS Users

Non-RFUMS Users

LSR II and Facscalibur

LSR II and Facscalibur

Assisted - $54/hr

Assisted - $108/hr

Unassisted - $33/hr

Unassisted - $66/hr

 

 

Sorting (ARIA)

Sorting (ARIA)

Set-up - $77/hr

Set-up - $155/hr

Assisted - $77/hr

Assisted - $155/hr

Unassisted - $62/hr

Unassisted - $124/hr

*Based on 15 minute intervals

All unassigned users must be trained and/or authorized by facility staff.

Training is available.

 

CONTACT

Department of Microbiology and Immunology
Robert Dickinson, Flow Cytometry Manager
Phone (847) 578-3000 x 3554
Room 2.218
E-mail: robert.dickinson@rosalindfranklin.edu
 

Alternate Contacts
David N. Everly, Jr., Ph.D.
Phone: (847) 578-3000 x 8653 
Room 2.251
E-mail: david.everly@rosalindfranklin.edu

Joseph Reynolds, Ph.D.
Phone: (847) 578-3000 x 8332 
Room 2.351
E-mail: joseph.reynolds@rosalindfranklin.edu

 

Last Updated: 11.24.2014

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