Flow Cytometry Core Facility

cytometry

About Our Facility

FACSAria

Creighton's FACSAria is a state of the art, 13-color, 3-laser system. 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.

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 Paramters

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 (280-1841 or  gperry@creighton.edu).

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