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Flow Cytometry Facility (Wistar)

Directors: Faust, Jeffrey S., MBA; Montaner, Luis J., DVM, DPhil

Summary:

Overview

The Flow Cytometry Shared Resource provides investigators with the technological resources and professional assistance for high quality, multiparameter flow cytometry analyses and sorting. The Facility is capable of cell sorting (sterile, at speeds up to 30,000 cells/sec) from homogeneous or mixed cell populations based on up to 32 fluorochromes, sorting up to six separate populations simultaneously, including human-derived samples at BSL-2 level. Facility personnel aid investigators in creating efficient and cost-effective experimental designs, through optimizing cytometry-specific reagent and fluorochrome selection, and offer assistance in operation of analysis instruments. Technical support is also provided for analyses of flow and imaging cytometry data for publication, presentation, and inclusion in grant applications, management of cytometric data (storage, archiving, and retrieval), and management of a site license for low-cost post-acquisition analysis software.

Affiliations:

People:

Resources:

Instruments

  • Amnis ImageStream flow cytometer ( Flow cytometer analyzer )

    ImageStream "produces multiple high resolution images of every cell directly in flow, including brightfield, darkfield (SSC) and up to 10 fluorescent markers with sensitivity exceeding conventional flow cytometers."

    The Amnis ImageStream is operated by facility personnel only.

  • FACSAriaII high-speed cell sorter – 16 color, 4 lasers ( Flow cytometer sorter )

    This sorter is capable of sorting up to 4 subpopulations simultaneously at rates up to 25,000 cells/second (70u). Higher threshold rates of up to 70,000 events per second can be achieved without affecting purity.

    • Four lasers:
    -- UV (375 nm)
    -- Violet (405 nm)
    -- Blue (488 nm)
    -- Red (633 nm)
    • 16-color cell sorter

    This instrument is operated by facility personnel only.

  • FACSCalibur analyzer – 4 color, 2 lasers ( Flow cytometer analyzer )

    • 4 color
    • Capable of user-friendly analysis
    • Two lasers:
    — blue (488 nm)
    — red (635 nm)
    • Low-speed (up to 5,000 events per second) analysis-only instrument

  • FACSCelesta analyzer with high-throughput sampler – 12 color, 3 lasers ( Flow cytometer analyzer )

    • 12 color
    • Capable of investigator-operated analysis
    • Three laser
    -- Violet (405 nm)
    -- Blue (488 nm)
    -- Red (640 nm)

  • FACSMelody user-friendly cell sorter – 9 color, 3 lasers ( Flow cytometer sorter )

    This sorter is capable of sorting at rates up to 10,000 cells/second.
    • Three lasers:
    -- Violet (405 nm)
    -- Blue (488 nm)
    -- Red (642 nm)
    • 9-color sorter

    This instrument is operated by facility personnel only.

  • FACSymphony analyzer with high-throughput sampler – 28 color, 5 lasers ( Flow cytometer analyzer )

    • 28 color
    • Capable of user friendly analysis
    • Five lasers:
    -- UV (355 nm)
    -- blue (488 nm)
    -- red (637 nm)
    -- violet (405 nm)
    -- yellow green (561 nm)
    • High-speed analysis-only instrument

  • Flow cytometry data analysis workstations ( Computer workstation )

    PC and Mac workstations for post-acquisition analysis.

  • LSRII analyzer – 14 color, 4 lasers ( Flow cytometer analyzer )

    • 14-color
    • Capable of user-friendly analysis
    • Four lasers:
    — blue (488 nm)
    — red (635 nm)
    — green (532 nm)
    — violet (405 nm)
    • High-speed analysis-only instrument

  • LSRII analyzer – 18 color, 4 lasers ( Flow cytometer analyzer )

    • 18-color
    • Capable of user-friendly analysis
    • Four lasers:
    — blue (488 nm)
    — red (635 nm)
    — green (532 nm)
    — violet (405 nm)
    • High-speed analysis-only instrument

  • MoFlo Astrios high-speed cell sorter – 32 color, 7 lasers ( Flow cytometer sorter )

    This sorter is capable of sorting six different subpopulations at the same time at rates up to 25,000 cells/second (70u). According to the technical specification it can achieve a validated sort rate of up to 70,000 events per second .

    • Seven lasers:
    -- UV (355 nm)
    -- Violet (405 nm)
    -- Blue (488 nm)
    -- Green (532 nm)
    -- Yellow Green (561 nm)
    -- Orange (592 nm)
    -- Red (642 nm)
    • 32-color sorter

    This instrument is operated by facility personnel only.

Services

  • BSL2+ biohazardous cell sorting ( Material processing service )

    "BSL2+ level flow cytometry analysis and sorting of potentially infectious biological materials, utilizing the BD ARIAII. This provides an in-house resource for running non-characterized human-derived material, infected cell lines, or other potentially bio-hazardous samples."

  • Cell-cycle analysis ( Material analysis service )

    "Cell-cycle analysis with propidium iodide or 7-AAD, anti-BrDU antibodies, tracking of cell division using CFSE or similar dyes, apoptosis in cell lines and primary cell populations using various techniques, including propidium iodide (PI) staining, terminal desoxytransferase (TdT) staining, and Annexin V labeling."

  • Flow cytometer access ( Access service )

    User operated flow cytometry analysis.

  • Flow cytometry analysis by facility operators ( Material analysis service )

    Includes post-acquisition analysis of acquired data

  • Flow cytometry collaboration facilitation ( Support service )

    "Facilitating collaborative efforts among Wistar investigators, and where possible, with other Institute shared resources and other cytometry resources. Resource operators are familiar with technical aspects of many of the investigators’ experiments, allowing them to facilitate sharing of reagents and techniques and to encourage collaborations among Wistar users. Mr. Faust, manager, is well acquainted with a vast external network of flow cytometry laboratories and researchers in the field, and able to provide additional opportunities for collaborative sharing and development of techniques. Additionally, as part of the Shared Resources Committee, he interacts on a regular basis with Wistar Shared Resource directors and is well-informed of resources available that may be used in conjunction with flow cytometry to augment or enhance research projects."

  • Flow cytometry data management service ( Data storage service )

    "Storing, archiving, and retrieving flow cytometric data and disseminating it through a local network and communication with the Institute network servers; working together with the Institute Information Systems to provide 24 hour accessibility and data security."

  • Flow cytometry Training and Consultation service ( Support service )

    "Consulting with investigators to determine individual flow cytometry needs; to aid in development of appropriate and cost-effective protocols and techniques. Resource staff assists in design of large multicolor fluorochromes panels and setting of compensation matrices, and provide advice on standard as well as specialized services, e.g. whether to use common reagents for surface phenotyping or specialized reagents and new techniques. Training of investigators on “user-friendly” flow cytometry instrumentation."

  • Flow cytometry training and ongoing advice ( Training service )

    "Training and providing ongoing advice to investigators in the use of investigator-operated instrumentation so they may perform data acquisition and analysis without hands-on operator assistance; providing technical support for analysis of flow cytometry/cell sorting data for publication, presentation, and inclusion in grant applications. Staff is conveniently located in order to provide immediate assistance and advice to investigators on user-friendly instruments."

  • Fluorescent protein expression analysis ( Material analysis service )

    "Fluorescent protein expression with GFP, YFP, CFP, and various red fluorescent proteins including monomeric red proteins such as Cherry."

  • Functional assay service ( Material analysis service )

    "Functional assays, such as intercellular conjugation run on cells stained with intracellular calcein, hydroxyethidine, or PKH26, as well as surface markers; oxidative stress; pH; calcium flux. Total cellular protein can be measured with FITC or SR101."

  • Immunophenotyping ( Material analysis service )

    "Immunophenotyping/immunofluorescence utilizing single or multiple laser analysis of up to 18 fluorochromes combined with forward- and right angle-light scatter. This includes various immunofluorescence analyses such as cell surface and intracellular phenotyping and making use of QuantumDot (QDot) technology."

  • Non-infectious cell sorting ( Material processing service )

    "Cell sorting (operator-assisted) of up to 4 separate populations using up to 10 fluorochromes on the high-speed BD ARIAII or 6 fluorochromes on the MoFlo instrument. High purity, high recovery, sterile cell sorting (single or multiple laser) may be performed on any cell population detectable by analysis (above). Up to four populations may be collected simultaneously, at sorting rates of up to 25,000 cells/second (90 million/hour)."

Software

  • FlowJo ( Software )

    "FlowJo should be the first step in the process of analyzing flow cytometry data. FlowJo can analyze data generated by any flow cytometer from any manufacturer. FlowJo has a number of different analysis platforms that let you not only perform standard analyses such as gating and statistics, but also specialized analyses such as DNA/Cell Cycle, Kinetics (Calcium flux), Proliferation, Calibration, and Statistical Comparison. You may find that FlowJo's sophisticated tools for generating output (graphical or tabular) are sufficient for you to generate publication-quality material.... but if not, FlowJo makes it easy for you to copy any graphs, statistics, or other information into other programs for further analysis and presentation."


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Last updated: 2020-03-16T11:35:58.847-04:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016