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University of Utah Core Flow Cytometry Facility

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University of Utah HSC Core Research Facilities

Flow Cytometry Facility*

The Flow Cytometry Core Facility offers quantitative, multiparameter florescence analysis and cell sorting services to the research community. Becton-Dickinson analyzers include two modified FACScans (two lasers, 5 fluorescence detectors) which are available for user operation or core-operated service. One of these FACScans is located at HCI in room 4274 (in the open lab space) and the other is in the Core's Analyzer Lab in the Wintrobe Building.

A FACSCanto-II (three lasers, 8 fluorescence detectors) is also located in the Core's Analyzer Lab. The Canto is also equipped with a High Throughput Sampler (HTS) designed to draw sample directly from the wells of a microtiter plate. The HTS is ideal for cytometric bead array applications which are set up in microtiter plates.

Analyzers are available to trained users after hours and on weekends with access to the Analyzer Lab via one's university ID card. Cell sorting services are provided by the core staff on a FACSAria-II SORP high speed cell sorter which has five excitation lasers (UV, 405nm, 488nm, 563nm and 635nm) and 13 fluorescence detectors. Sorted cells may be collected in bulk (tubes) or deposited in defined numbers of cells/well in a microtiter or other culture plate. Flow cytometry-specific data analysis software is available for the on-line analysis of data files by users or core staff.

Instrumentation

Upgraded 5-Color FACScan Analyzers (2 available):

  • A bench top analyzer with two lasers for fluorochrome excitation: The primary laser is a 15 mw argon (488 nm) laser and the secondary laser is a 25 mW red diode (637 nm) laser. One instrument is located in room 603 of the Wintrobe Building (Analyzer Lab) and the second is located in a satellite lab on the third floor of the Huntsman Cancer Institute (bench 3-O).
  • Seven detectors: Two for light scatter (forward and 90°) and five for fluorescence. The optical filters for the fluorescence detectors are fixed at FL1 = 530±15 nm, FL2 = 585±21 nm, FL3 = 668 nm long pass, FL4 = 666±13 nm and FL5 = 740 nm long pass.
  • Suitable for immunofluorescence (up to 5 color) and DNA content/cell cycle analysis. A link to a pdf covering basics of instrument operation is available below.
  • Commonly used fluorochromes (please note that Texas Red can not be used as a single fluorochrome on these instruments):

   

Download "Flurochrome Guide"pdf

Download "FACScan Operation Guidlines" pdf

FACSCanto-II Analyzer:

  • A new digital bench top analyzer with three lasersfor fluorochrome excitation: The primary laser is a 20 mw argon (488 nm) laser and the secondary lasers are a 17 mW red diode (633 nm) laser and a 30 mW violet diode (405 nm) laser. This instrument is located in room 603 of the Wintrobe Building (Analyzer Lab).
  • Ten detectors: Two for light scatter (forward and 90°) and eight for fluorescence arranged for four on the blue 488 nm laser, two on the red 633 nm laser and two on the violet 405 nm laser (4-2-2 configuration). The optical filters can be changed but the basic configuration is:

For the blue 488 nm laser;

1. Detector A = 750-810 nm(PE-Cy7)

2. Detector B = 670 nm long pass (PerCP, PerCP-Cy5.5)

3. Detector D = 564-606 nm (PE)

4. Detector E = 515-545 nm (FITC, GFP)

For the red 633 nm laser;

1. Detector A = 750-810 nm (APC-Cy7, APC-H7)

2. Detector C = 650-670 nm (APC, Alexa-633, Cy5)

For the violet 405 nm laser;

1. Detector A = 502-535 nm (AmCyan)

2. Detector B = 425-475 nm (Pacific Blue, DAPI, Cascade Blue).

  • The Canto can be used with the fluorochromes outlined above for the 5-color FACScans plus those excited by the violet laser listed in the preceding paragraph.
  • Additionally, the Canto has a High Throughput Sampler (HTS) which is a robotic option to draw samples directly from a 96-well microtiter plate. This option is ideal for those who stain cells in microtiter plates or who are doing cytometric bead arrays in microtiter plates.

FACSVantage SE High Speed Cell Sorter:

  • A dual laser instrument (argon and helium-neon lasers) capable of generating three simultaneous laser beams for fluorochrome excitation (UV at 340-360nm, blue at 488nm and red at 633nm). Please note that Texas Red can not be used as a fluorochrome with these lasers! Also, please be aware that PerCP is a very weak fluorochrome on a jet-in-air sorter like the Vantage SE and should not be used unless you are working with a very brightly staining marker. Instead of PerCP you can use the tandem dye PerCP-Cy5.5 which was specifically designed as an FL3 fluorochrome for jet-in-air cell sorters.
  • Ten detectors: Two for light scatter (forward and 90°)and eight for fluorescence. The fluorescence detectors are arranged so that four receive signals from the primary 488 nm beam, two from the second 633 nm beam and two from the third 340-360 nm beam. Optical filters may be changed on each of these fluorescence detectors to capture a particular fluorescence emission wavelength. A maximum of six fluorescent signals can be collected at one time.
  • Cell sorting capabilities for two populations defined by any combination of light scatter and fluorescence characteristics. The maximum cell analysis rate for sorting is 8 x 106 cells/hour. To view more information about cell sorting click here.
  • A recirculation water bath is available to maintain the sample tube and the sorted cells at a defined temperature.
  • An Automatic Cell Deposition Unit (ACDU) option allows the sorting of defined numbers of cells into the wells of a microtiter or other tissue culture plate.

Support Services:

Full service by lab personnel is available for staining, analyzing and data analysis during normal working hours. Training is provided for individuals who wish to operate the FACScans themselves and take advantage of off-hour access. Collaborative support is also available for developing new assays and applications of the technology.