This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Modular Flow (MoFlo) high-speed cell sorter (DakoCytomation;Fort Collins, CO) is designed to sort at 70,000 events/second, with 99% purity. The MoFlo is configured with three lasers, equipped with the capability of sorting cells into microtiter plates. Also included are a water-cooled Coherent Innova 90-C Argon ion 350nm (UV) laser, an air-cooled Spectraphysics Argon ion 488nm (blue) laser, and a Melles-Griot 633nm (red) Helium-Neon laser. This configuration provides the capability of detecting up to six colors, and the software configuration allows for up to four color immunophenotyping simultaneously using the following fluorochromes: FITC, PE, PE-Cy-7, and APC. Additionally, the instrument is equipped with a CyClone Automated Cell Deposition Unit, which allows for rapid and accurate cell deposition into various multi-well plate formats (single cell to multiple cells per well). The MoFlo is openly reconfigurable and has additional expansion capabilities for up to four additional detectors, should the needs of our research shift. As an added safety feature, an E-Vac (aerosol evacuation system) is installed to contain aerosols produced during sorting. Flow Cytometry Facility: This facility is a component of the Department of Pathology and Laboratory Medicine at Roger Williams Hospital. As such it is certified by the College of American Pathologists (CAP), and is CLIA 88 compliant. The facility is housed in an 800 square foot area and is maintained and renovated as needed by the hospital. The facility operates at a level two biohazard status, and Universal Precautions are strictly adhered to. There are two full time technologists available for assistance. FACSCalibur: The FACSCalibur (Becton Dickinson) is equipped with a loader (FACS loader) for automatic acquisition of samples. It also has a FACStation for analysis. Analysis programs include: FACScomp, SimulSET, MultiSET, Attractors, CELLQuest, and ModFit LT. LSRII flow cytometer: An LSRII flow cytometer (Becton Dickinson) is also available for immunophenotyping analysis. In contrast to the MoFlo cell sorter, which has a dedicated operator, COBRE personnel are encouraged to train on the LSRII, which will allow them independent use of the instrument. Training is provided by facility personnel, who will ensure that they are proficient in operating the facility equipment.
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