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
Single cell sort and index sorting
Spatially separated lasers.
– 488nm 50mW
– 405nm 50mW
– 561nm 50mW
– 640nm 40mW
Optical configuration: AriaII
Single cell sort and index sorting
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.
How are the sorters named?
The nomenclature reflects the instruments as listed in the CAT Facility iLab’s site: Model_# lasers-# detectors.
How do I decide which sorter to use?
- If you are just looking for an overview of which fluorophores can be used on each sorter, see the Sorter Comparison Table (also found in the button before the Sorter FAQ section).
- If you need further assistance, please contact CATHelp@bsd.uchicago.edu and the staff will help you decide which analyzer is best for your experiment.
What is the configuration of the sorters?
This information can be found here and the button above the Sorter FAQs section:
This table contains the laser lines and bandpass filters for all of our sorters. It also contains a list of popular fluorophores that can be assigned to each detector. Only one fluorophore per row should be used in a panel of antibodies, if you choose two or more fluorophores from a row you will not be able to distinguish them.
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.
- We will assume that you will be dropping off samples for the staff to run unless you have discussed/completed sorter training with the staff.
- 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. However 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
Aria Normal Operating State
If you schedule a sorter we will assume that you will be dropping off samples for the staff to run. If you plan on sorting frequently after hours you may request to be trained to run the sorters unassisted. More information on training can be found here.
- 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.
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.
Using the Sorters
How should I prepare my samples for my experiment?
- 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.
- 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)
- FACS buffer – can be cell dependent
Cell Concentration and Volume
- 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.
- For 5 mL flow tubes please use Falcon #352008
- 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.
What should I bring to collect my sorted cells?
- 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
- Trizol LS (more concentrated, recommended) ThermoFisher #10296010
- Trizol ThermoFisher #15596026
- 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.
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.
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 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.
Assisted Cell SortingAria
- Charge as booked on iLab
- Booked unused time charged in full
Unassisted Cell SortingAria
- Charged as booked on iLab
- Discount applies outside of normal operating hours only
Unassisted Cell SortingMACSQuant Tyto
- Charged based on time used
- Staff time will be charged for initial template settings (15min at $28/hr). Once a template is designed, the instrument can be used without involvement from the staff
- Cartridges available from the CAT Facility at a discounted rate ($100/unit)