The flow cytometry facility is contained and maintained within the Department of Molecular Sciences, MSB214, and the Cancer Research Building, Rm. 146. The facility includes two analytical flow cytometers and a cell sorter. The facility functions as a core laboratory accessible to all PIs and research laboratories at UTHSC as well as other institutions and companies in the Memphis area and is operated on a fee-for-service basis. The facility is directed by Dr. Tony Marion, Professor, Department of Molecular Sciences and has a full-time, highly trained operator, Dr. Dan Rosson. Dr. Rosson has many years of training and expertise in cell biology, particularly cancer cell biology, and flow cytometry. Both Drs. Rosson and Marion are available to all users for help in designing and implementing protocols to implement flow cytometry and cell sorting within their research projects.
FACSAria Special Order System (BD Biosciences)
The cell sorter is equipped with four lasers with twelve fluorescence detectors, in addition to FSC and SSC PMTs. The 0-100 mW 488 nm blue diode laser is equipped with five fluorescence detectors and a SSC detector in an octagon configuration. This laser is filtered for detection of FITC, PE, PerCP-Cy5.5, PE-TxRd, and PE-Cy7. The 30 mW 638 nm red diode laser is equipped with three fluorescence detectors in a trigon configuration, and is filtered for detection of APC, APC-Cy7, and Alexa700. The 50 mW 405 nm violet diode laser has two fluorescence detectors in a trigon configuration, and is filtered for detection of Pacific Blue/Cascade Blue/Alexa405 and AmCyan/Pacific Orange/Alexa430. The 20 mW 355 nm solid-state UV laser has two fluorescence detectors in an octagon configuration, and has upgrade capacity for Qdots. Detection of Hoechst, Indo-1 (Ca-bound and unbound), and DAPI can be accomplished using this laser. FSC detection is selectable between PMT (for increased sensitivity and detection of subcellular particles and bacteria < 0.2 micrometer) and diode (for eukaryotic cells). The instrument has two-and four-way sort capacity (into tubes or microtubes) with sort rates of 22,000 events/sec in purity sort mode with predicted recovery of 80% and 98% purity. This sort rate allows for sorting the entire mononuclear cell population from a single mouse spleen in approximately 1 hour. The unit is also specially equipped with: 1) temperature controlled sample injection and cell collection chambers, 2) biosafety level-2 aerosol containment (for sorting infectious samples), and 3) automated cell deposition (including single cell) into microwell plates or onto microscope slides.
LSR II Special Option Flow Cytometer (BD Biosciences)
The LSR II flow cytometer is equipped with four lasers with eleven fluorescence detectors, in addition to FSC and SSC PMTs. The 22 mW 488 nm blue laser is equipped with four fluorescence detectors and a SSC PMT, in an octagon configuration, and is filtered for detection of FITC, PE, PerCP-Cy5.5, PE-TxRd, and PE-Cy7. The 20 mW 638 nm red laser has three fluorescence detectors (in a trigon configuration) which are capable of detecting APC, APC-Cy7, and Alexa700. The 22 mW 405 nm violet laser has two fluorescence detectors (in a trigon configuration), that have capacity to detect Pacific Blue/Cascade blue/Alexa405, and AmCyan/Pacific Orange/Alexa430. The 60 mW 355 nm solid-state UV laser has two fluorescence detectors (in a trigon configuration), which can detect Hoechst, Indo-1 (Ca-bound and unbound) for Ca2+-flux measurements, and DAPI. This instrument is also equipped with a programmable HTS (high throughput screening device) for automated collection of data from 96-384 well microplates.
FACSCalibur (Sort) (BD Biosciences)
The FACSCalibur flow cytometer is equipped with two lasers, a 488 nm blue and 635 nm red, and four fluorescence detectors in addition to forward and side scatter. The instrument will be located in the recently completed Cancer Biology Building. This instrument has limited sorting capability but because of the relatively slow sort speed is used primarily for analysis.