Cytometry and Antibody Technology

Model: BD FACSAria Fusion
Manufacturer: BD Biosciences

Priority to primary patient samples: The CAT Facility recognize the rarity and importance of primary patient samples. We will give priority to users bringing patients samples to the CAT Facility regardless of the schedule. Call the CAT Facility to let us know.

The Aria Fusion 5-18 is the only cell sorter equipped to protect the operator from biohazards. As such, it is mainly reserved for BSL2 material. Mouse sample and non-infectious cell lines should be processed on the other cell sorters.

Spatially separated lasers.
– 488nm 100mW
– 405nm 100mW
– 561nm 100mW
– 640nm 100mW
– 355nm (UV) 15mW

Optical configuration: Aria Fusion

Encased in laminar flow hood for biohazard protection
Plate sort
Single cell sort and index sorting

Model: BD FACSAria III
Manufacturer: BD Biosciences

Spatially separated lasers.
– 488nm 50mW
– 405nm 50mW
– 561nm 50mW
– 640nm 15mW

Optical configuration: AriaIIIu

Plate sort not available on this instrument

Model: BD FACSAria II
Manufacturer: BD Biosciences

Spatially separated lasers.
– 488nm 50mW
– 405nm 50mW
– 561nm 50mW
– 640nm 40mW

Optical configuration: AriaII

Plate sort
Single cell sort and index sorting

Model: MACSQuant Tyto
Manufacturer: Miltenyi Biotec

Sort using single use cartridge to ensure complete sterility and biosafety. Bulk sort of 1 population at a time, flow through is collected and can be sorted again.

Spatially separated lasers, 8 fluorophore detectors available.
488nm: 525/50, 585/40, 693/38 , 750LP
405nm: 450/50, 510/50
635nm: 693/38, 750LP

Cartridges available directly from the CAT Facility at a discounted price.

See here for more information

Sorter FAQ

Aria Normal Operating State
  • Sheath: Leinco’s Clear Sort 10X sterile solution mixed with Milli-Q water
  • By default, the water bath is turned off. If you would like your sorted samples to be kept at a certain temperature please contact your operator to have the water bath turned on at your desired temperature
  • Upon user arrival, the instrument will be ready to go
    • CST performed
    • Drop Delay calculation completed
    • Probe cleaned with bleached
  • Self-sorting users may have to get the instrument started on their own.
Information About Materials
  • FACS buffer – can be cell dependent
    • 1X Ca/Mg2+ free PBS (HBSS also works)
    • Either FBS (0.5-5%) or BSA (0.1-1%)
    • Optional: EDTA (0.5-5mM) if you have very sticky cells
    • Commonly used: 1% FBS in 1X PBS (we strongly recommend that you filter FBS or the whole buffer to remove debris form the FBS)
    • We recommend bringing your sample in FACS buffer made with PBS or HBSS because it is similar to the sheath buffer in the sorters. If you bring cells in cell culture media instead of FACS buffer, we don’t recommend anything with phenol red as it might interfere with the autofluorescence. Collection tubes can contain whatever is best for your experiment.
  • 5 mL flow tubes: Falcon #352008
  • Trizol ThermoFisher #15596026
  • Trizol LS (more concentrated) ThermoFisher #10296010
Notes about Patient Samples
Patient samples are prioritized on the Aria Fusion

  • The CAT Facility recognizes the importance of samples coming from clinical settings. The CAT Facility will give priority to researchers with such a sample. Call the CAT Facility as soon as you know a sample will be made available to you so we can coordinate our efforts and find time in the schedule.
How do I schedule the sorters?
  • If you have not signed up for iLab, sign up there first. A video explaining how to use the iLab scheduler can be found here.
  • You can sign up for any of the three sorters -the Aria II, Aria IIIu, or Aria Fusion. The BD Aria Fusion is reserved for biohazardous material such as human samples. If your experiment does not require BSL2 material, it may be moved to another sorter. You will be notified in such a case.
  • The configurations of the sorters can be found on iLab and here.
  • When you sign up through iLab you will be asked to fill out a form to help the staff know what to set up
  • Once you have made a reservation, it will appear in Orange on the iLab scheduler. This means that the CAT Facility staff needs to review your sort information. If there is a problem, you will be contacted. But once the sort is booked, the time on the instrument is yours, review or not.
  • If you have any specific questions, please contact the staff at CATHelp@bsd.uchicago.edu with any quick questions or set up an appointment to go over your experiment setup
What should I bring to my appointment?
  1. Sample
    • Your cells can be brought in whatever media will keep them in good condition. We recommend FACS buffer (see information about materials above) made with PBS or HBSS because it is similar to the sheath buffer in the sorters. If you bring cells in media, we don’t recommend anything with phenol red as it might interfere with the autofluorescence.
    • Depending on the nozzle tip used, the cell concentration will vary
      • 70um tip (lymphocytes, splenocytes, small cells): 20-25 million cells per mL
      • 100um tip (most cell culture, lung and liver cells, etc): 10-15million cells per mL
      • At these concentrations, we can sort
        • 70um tip: 3mL/hr
        • 100um tip: 2mL/hr
      • These concentrations will offer an optimal sorting efficiency without impacting sort purity
      • The minimum recommended volume is 500uL.
      • Use 5mL or 15mL tubes only. Other tubes will not fit the sample chamber.
  2. Controls
    • For multi-parameter experiments, appropriate single cell controls are highly recommended
    • Gating controls, such as unstained cells or FMOs are needed to gate the desired population properly.
    • Use 5mL or 15mL tubes only. Other tubes will not fit the sample chamber.
  3. Collection tubes
    • The collection tube will vary with the expected results of your experiment and the downstream application. If you are unsure of which collection tubes to select, please contact CATHelp@bsd.uchicago.edu prior to your sort.
    • A wide collection of receptacles can be used, including:
      • 15mL tubes (sort 2 populations simultaneously)
      • 5mL tubes (sort 4 populations simultaneously)
      • Eppendorf tubes (sort 4 populations simultaneously)
      • Plates (6-well plates to 384 well plates) -> Available on the AriaII and Aria Fusion only
      • Other – contact the CAT facility staff if you have a non-traditional idea in mind or if you want to sort more than 4 populations from your sample.
    • The tube should contain collection media to preserve cell viability. 1X Ca/Mg2+ free PBS with either FBS (0.5-5%) or BSA (0.1-1%), HEPES buffered (10-25mM of HEPES, pH range 6.8 – 8.2) is recommended.
    • Variations might include:
      • The addition of EDTA or DNAse to prevent cell clumping
      • Higher protein concentration to help viability – you can sort into 50-100% FBS because the sample will dilute the collection buffer (see below)
      • Culture media
      • Trizol for DNA or RNA extraction
    • The sorter will generate an extra amount of volume:
      • 70um tip: ~1mL/million cell sorted
      • 100um tip: ~3mL per million cells sorted
      • If you plan on sorting directly in lysis buffer, be mindful of the dilution impact of this extra volume
        • The buffer should be kept at a maximum of 10% of total lysis buffer, or 20-25% if using Trizol LS
        • An alternative is to spin your cells after the sort and resuspend directly in lysis buffer.
Tips for Culturing Sorted Cells
NOTE: These tips are for the Arias. For true sterile sorting, you may want to consider the Tyto. Please contact the staff in advance to discuss your Tyto sort with the staff – you may need to alter your panel for the Tyto.

  • The Arias are kept as clean as we can, but should not be considered sterile instruments.
    • They use sterile 10X ClearSort Sheath mixed with MilliQ water
    • The sample probe is bleached between every experiment
    • The instruments are flushed with ETOH at the end of every day
    • The sheath lines are bleached on a weekly basis or as needed
    • Contamination spot tests are taken on all of the instruments on a weekly basis so the staff should be able to inform you of any issue
    • The in-line sheath filter is replaced every 6 months or as needed
  • The use of antibiotics is strongly recommended. Gentamycin has been shown to be more efficient than Pen/Strep
  • We recommend contacting the staff whenever the sorted cells are to be put back in culture. The staff will inform you of any specific issue regarding potential contaminations and will take every step needed to keep the instrument clean.
  • Should a sort get contaminated, inform the CAT Facility as soon as possible. It will allow the staff to take the appropriate action needed in a timely matter
What should I do at the time of my appointment?
NOTE: For COVID-specifc procedures, see our RRP.

  • The CAT Facility operator will be waiting for you at the instrument.
  • Your controls will be acquired and gates will be set based on your directions
  • A purity check will be run on your samples unless you direct otherwise
  • You will need to stay until the gates are set and the purity check is complete. Once the sort is under way, you will be able to leave the facility.
  • The Operator will email you at the end of the sort. This may occur when there is no sample left or if the is no more time available. In this case, the operator will present available solutions to complete the experiment.
What if there are problems with my sort?
  • If you encounter any problems with your sort, please make sure you communicate with the staff. We can’t help you if you don’t let us know. Again, the email is CATHelp@bsd.uchicago.edu
  • Cancellation policy: The sorters are billed on the amount of time that was booked or used, whichever is greater. Sorting reservation can be cancelled by the user on iLab up until the point where it has been approved by the core. At that point, you will need to contact the staff to cancel your time. Unused sort time will be billed in full.