Flow Cytometry /Cell sorter

The Flow Cytometry Core Facility is operated by the Microbiology and Immunology discipline.

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

Cytek

BD Biosciences

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.

bioBubble Class I Benchtop Biocontainment Enclosure (BBE)

The ARIA II cell sorter 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 ARIA II in the BBE allows for sorting primary human samples and other materials that are handled using BSL2 precautions.

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.

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

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

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

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