Core Center Resources:
Flow Cytometry
Flow Cytometry – Jianke Zhang, Ph.D., KCC, TJU
The Flow Cytometry Shared Resource (FCSR) is an established Kimmel Cancer Center (KCC) shared resource. It is located in a dedicated laboratory space of about 600 sq ft (room 606) on the 6th floor of the Bluemle Life Sciences Building, which allows Cancer Center members convenient access to this highly utilized service. The FCSR is the only shared resource that provides access to all Thomas Jefferson University investigators. The mission of the shared resource is to provide high quality, efficient and affordable analytical cytometry services to the KCC members in order to serve the basic science and the translational programs. The FCSR has been providing 1) the means for single and multi-parameter analysis and purification of cells from homogeneous or mixed cell populations, and 2) expertise in helping investigators to choose experimental conditions and reagents to optimize their use of the shared resource for their experimental needs. A Coulter EPICS Elite sorter was recently moved to a Biosafety Level (BSL)3 facility on the 7th floor in Bluemle Life Sciences Building in order to develop services for analysis and sorting of pathogen-infected cells
Equipment
- Cytomation MoFlo high-speed cell sorter: This instrument was purchased two years ago using a NIH equipment grant supplemented by institutional funds. It is capable of sorting up to 70,000 particles per second, and has an air-cooled 488 argon laser and a 633 Helium-Neon laser. It allows analysis and sorting based on up to 7-color immunofluorescence detection combined with analysis of forward- and right angle-light scatter.
- Coulter EliteESP Cell Sorter: The Enhanced Sensitivity Performance (ESP) Flow Cytometer is capable of sorting between 5,000 and 15,000 particles per second (defined as high speed). The flow cytometer is equipped with 3 lasers, including a Coherent Innova 305 water-cooled Argon laser, an air-cooled Argon laser (488nm), and an air-cooled Helium-Neon laser (633nm). This instrument has a gated amplifier, 5 photomultiplier tubes (PMT, photon detectors to collect light emission), a Cytek Time Zero Module, and a temperature regulation unit. The computer workstation is connected to the network and is a Coulter Flowcentre2, Pentium III, 550Mhz/128MB RAM/12GB HD/CD-ROM, Yamaha CD-RW CD-ROM writer, external 100MB Zip drive, with a HP2000C photo-quality inkjet printer.
- Coulter XL-MCL Automated Flow Cytometer: The Coulter XL-MCL is an automated cytometry system designed for high-throughput qualitative and quantitative measurements of biological and physical properties of cells and other particles. This system was purchased with institutional funds in November 1999, and has been operative since 12/7/99. The XL-MCL is equipped with one 488 nm aircooled Argon laser and 5 PMTs (as well as forward scatter measure) allowing multiple parameter analysis. The system includes an automated carousel sample loader, which allows for “hand-free” analysis of up to 32 samples per carousel. In addition, the XL-MCL is a user-friendly instrument, allowing investigators, after appropriate training, to analyze their own samples manually. The computer workstation is connected to the network and is a Coulter Flowcentre, Pentium II, 200MHz/32MB RAM/4GB HD/CD-ROM, internal 100MB Zip drive, with a Lexmark OptraM410n laser printer.
- Becton-Dickinson FACSCalibur Benchtop Analyzer: The FACSCalibur bench top analyzer is equipped with an air-cooled Argon laser, an air-cooled Helium-Neon laser, and five photomultiplier tubes. The computer workstation is connected to the network and is a Becton Dickinson FACStation. The analyses capabilities of this instrument are similar to those of the Coulter XL-MCL, but utilize a different platform, allowing for enhanced flexibility within the shared resource.
- Becton-Dickinson FACScan Benchtop Analyzer has an air-cooled Argon laser, and a temperature regulation unit. Analysis is based on up to three-color immunofluorescence and light scatter characteristics.
- Workstation and software for extended data analysis: The shared resource has a network-connected computer workstation for off-line reanalysis of stored data. This system is a Celeron 400Mhz/64MB RAM/13GB HD/CD-ROM, loaded with various cytometry programs including FloJo, EXPO2, MulticycleAV, Multitime, MFI, and WinMDI.
Flow Cytometry – Bruce Boman, M.D., Ph.D., Christiana Care
Establishment of a flow cytometry facility is planned for 2009 in conjunction with opening of the new Pavilion at HFGCC. The Pavilion will be home to the Center for Translational Cancer Research (CTCR) and other oncology programs. The new building, scheduled to open in summer 2009, includes four stories and 124,000 square feet of space connected to the existing 60,000-square-foot HFGCC. The flow cytometry facility is being funded by The Friends of the Helen F. Graham Cancer Center through a special fundraising event planned on May 15, 2009. Previous fundraising events by the Friends over the past six years have provided more than $1,000,000 of philanthropy for Christiana Care’s cancer program.
The flow cytometry facility will provide state-of-the-art fluorescence-activated cell sorting and analysis, including multi-color cell surface phenotyping, detection of intracellular and cell surface molecules, studies of cell proliferation such as cell cycle analysis and apoptosis, and isolation of cell subpopulations expressing specific markers such as stem-cell markers. The shared resource allows for reliable and accurate characterization of a wide spectrum of biological samples and provides services that can be tailored to experimental designs in a variety of fields toward the understanding of the biology and treatment of cancer and other disease processes.
The facility will house a BD FACSAria™ cell sorter for high performance flow cytometry. This easy-to-use benchtop system delivers high-speed sorting and multicolor analysis. The BD FACSAria instrument is the first benchtop sorter that incorporates a fixed-alignment cuvette flow cell that provides superior fluorescence sensitivity. The fixed optical system offers freedom from instrument maintenance and alignment. Sort-setup and sort-monitoring features in the system software make the BD FACSAria instrument easy to use. BD FACS™ Accudrop technology is integrated into the system for quick and accurate drop-delay determination. Stream monitoring of the breakoff point and clog detection are incorporated for walkaway sorting.
Flow Cytometry – Robert Mason, Ph.D., Nemours
The Flow Cytometry/FACS facility provides tools to study, analyze, select and purify cells using endogenously produced fluorescent tags or antibodies to specific cell surface or intracellular markers. The Facility operates the Beckman-Coulter Altra Fluorescence Activated Cell Sorter (FACS). The advantage over other cell sorters, including those which are available at other Core facilities nearby at the University of Delaware, is that ours has lasers with more variation of wavelengths and the capability of high-speed sorting, which result in higher yields or viable cells. The Flow Cytometry/FACS Core staff is available to advise on study design, perform analyses, and sort cells into specific populations for additional study or culture. Current Flow Cytometry/FACS Core staff includes George R. Dodge, PhD, Flow Cytometry/FACS Core Director, and Joan Pugarelli, BS.