FACSAria-IIu

The Aria is the first benchtop sorter that incorporates a fixed-alignment cuvette flow cell. This feature gives the Aria excellent fluorescence sensitivity.  

Creighton's FACSAria was the first FACSAria in the world equipped with an air-cooled UV laser. In the past the optical bench of the standard FACSAria used fiber optic cables to route the laser beam from the laser to the cuvette flow cell.  Unfortunately, this precluded the use of UV lasers on the Aria, as the UV beam severely degraded the fiber optic cables. Larry Duckett at Becton Dickinson designed and built a completely new optical bench for us, allowing us to incorporate a UV laser. On our instrument, all of the laser beams are directed to the flow cell cuvette using standard optical mirrors and mirror mounts, thus eliminating the fiber optic cables and giving us flexibility for future upgrades.

Another optical refinement that we have made on our instrument is the inclusion of a forward scatter photomultiplier tube (PMT). Standard FACSArias only have a forward scatter (FSC) photo-diode, which is approximately 1-log less sensitive than the FSC-PMT. The addition of the FSC-PMT allows us significantly greater sensitivity for small particles like platelets and bacteria.

In addition to its superior optical characteristics, the FACSAria is a  high speed sorter. It can sort up to 4 populations simultaneously, at a rate of up to 30,000 cells/second. We have also included the "cloning" option, which allows us to sort any number of cells (from 1 cell to millions of cells) into any container. This includes microtiter plates, PCR wells, glass slides and petri dishes.

The FACSAria is running the DiVA (version 6) software from BD, and all data collected on the FACSAria are stored as listmode files (FCS3.0) on its Windows XP based computer.  All data is backed up onto an external hard drive on at least a weekly basis, and is also backed up to a secondary site on a quarterly basis. More recently, in March of 2019, an additional laser (561nm) was added to the instrument to expand analysis capabilities and increase flexibility for panel design.

 

FACSAria Specifications

Three Lasers for Excitation

  • 488nm Coherent Saphire (100mW)
  • 638nm Coherent Cube (30mW)
  • 355nm JDS-Uniphase/X-Cyte Lightwave (20mW)

Two Scatter Parameters

  • Forward Scatter Diode
  • Forward Scatter PMT
  • Side Scatter

 

    Thirteen Fluorescence Parameters

    7 off the blue laser (488nm)

    • 530nm - FITC, Alexa488, eGFP, CFSE, Syto9, Rhodamine123
    • 575nm - PE
    • 610nm - PE-TexasRed, ECD, Red-613, PI
    • 670nm - PerCP, PE-Cy5
    • 695nm - PerCP-Cy5.5
    • 780nm - PE-Cy7

    3 off the red laser (638nm)

    • 660nm - APC, Alexa647
    • 725nm - Alexa700
    • 780nm - APC-Cy7

    3 off the UV laser (355nm)

    • 440nm - Hoechst, DAPI, Indo-1 (bound)
    • 530nm - Indo-1 (free), QDot525
    • 585nm - QDot585
    • 675nm - Hoechst
    • 780nm - QDot800

    Fluorochromes Successfully Used On Our FACSAria

    The following list represents fluorochromes we have successfully used on our FACSAria.  There are other fluorochromes that will work, so it is by no means comprehensive or all inclusive.  It just represents fluorochromes that our users have chosen to use.  If you have a question about a specific fluorochrome, please ask Dr. Perry (402.280.1841 or gperry@creighton.edu).

    View fluorochromes

    AlexaFluor-488
    AlexaFluor-700
    Allophycocyanin (APC)
    7-Aminoactinomycin D (7-AAD)
    APC-Cy7
    CFSE (Carboxyfluorescein Sucinimidyl ester)
    CFDA (Carboxyfluorescein Diacetate)
    CyChrome (same ast PE-Cy5)
    DAPI
    DsRed
    ECD (same as PE-TexasRed)
    Ethidium Bromide (EB)
    FITC
    Fluo-3
    Fluo-4
    Green Fluorescent Protein (GFP, eGFP)
    Hoechst 33342
    Hoechst 33258
    Indo-1 AM
    PerCP
    PerCP-Cy5.5
    Phycoerythrin (PE)
    PE-TexasRed
    PE-Cy5
    PC5 (same as PE-Cy5)
    PE-Cy7
    Propidium Iodide (PI)
    Quantum Dots (QDots) - Q525, Q585, Q805
    Rhodamine 123
    Spectral Red
    Thiazole Orange

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