Flow Cytometry & Fluorescence Activated Cell Sorting Core

Appointments with staff are available Monday through Friday from 10am to 5pm.

Requests for cell sorting can be made using the online schedule or by contacting the FACS Staff by email at facs@pathology.wustl.edu. The online schedule is accessible only to Washington University researchers and requires a username and password.

To schedule cell sorting on the Aria II-1, go to the online schedule, choose the time you would like to begin and end, click on REQUEST and fill out the pop-up window with your lab and sort information. An email will be sent to the FACS staff detailing your request and your request will be added to the sorting calendar. You will receive a confirmation email when your request has been added to the schedule. 

If you do not receive a confirmation email, your sort has not been scheduled.

If the setup notes for your sorting do not indicate special requirements, the AriaII-1 will be set up with a default 85um nozzle @ 45psi. If your sort requires a special setup (i.e. changes in nozzle size, changes in pressure, single cell sorting into 96- or 384- well plates), please include this information in the notes section of the sorting request form and add 30 minutes to your requested sorting time to allow changes from the default setup (NOTE: you will be charged for this special setup time).

Trained operators will be able to reserve time directly on the AriaII-2 schedule. Training consists of two sessions in which modalities of sorting and instrument set ups are illustrated (see instructions and training section). People are encouraged to attend the two training sessions in a short time frame between each other. They are also encouraged to initially sort during working hours when FACS staff are present until they have acquired experience with troubleshooting and problems that may arise during sorting.

The AriaII-3 is a BSL2+ level sorter, as it is contained in a Bioprotector cabinet, which maximizes aerosol containment. Use of the BSL2+ sorter is encouraged for clinical samples when testing is uncertain and for cells infected with BSL2+ level pathogens. For sorting on Aria II-3 please contact the FACS staff at facs@pathology.wustl.edu.

Nozzles available to each sorter:

• 85um @ 45psi: default
• 70um @ 70psi: small/medium cell size
• 100um @ 20psi: very large cell sizes

New users are provided with training before being able to operate the instruments.

The online schedule is accessible only to trained users. To obtain training, please contact the FACS staff at facs@pathology.wustl.edu. Once trained, you will be issued a username and password to access the online schedule and an account will be set up on the instrument.

The training on the FACS analyzers is free of charge.

Training for cell sorting is provided only to users that are familiar with the Diva software and regularly operate the LSRII, Fortessa and or Canto analyzers. The training on the AriaII-2 User-operated Cell Sorter consists of two parts:

• Step 1: Introduction to instrument features and sort set ups. Fee: $40.00.
• Step 2: Supervised sorting. The trainees will sort their own cells under the supervision and with help of the trainer, if needed. Fee: $80.00.

Instrument fees are based on the following hourly rates billed in 15 minute increments for sorters and in 1 minute increments for analyzers. The total fee is calculated based on the booked hours plus any actual time used in excess of the booked hours. Please make sure that you delete time as soon as you know you will not be able to use it. This is important in order to grant everyone access to the machines.

FACSAriaII-1 cancellations

• No fee will be charged for the booked hours if the appointment is canceled 24 hours in advance.
• 50% of the hourly fee will be charged for unused booked time if the appointment is not canceled 24 hours in advance and if nobody requests the canceled time.

All sorters provide high-speed, high purity multi-parameter cell sorting into tubes (15ml, 5ml or 1.5ml) and are also capable of sorting directly into 96 or 384-well plates.

• BD FACSAriaII-1: Operated by the core staff only. Lasers: 355nm, 405nm, 488nm, 561nm, 633nm. Up to 14 colors detected. AriaII-1 Filter Configuration (pdf).
• BD FACSAriaII-2: Available for operation by trained users. Lasers: 405nm, 488nm, 633nm. Up to 10 colors detected. AriaII-2 Filter Configuration (pdf).
• BD FACSAriaII-3: Available in BSL2+. Recommended for sorting of infected animal cells, clinical samples when testing uncertain, yeast and bacteria. Long cleaning is required in specific circumstances. Lasers: 405nm, 488nm, 561nm, 633nm. Up to 11 colors detected. AriaII-3 Filter Configuration (pdf).

All BD FacsAriaII Cell Sorters have the BD Tm Aerosol Management Option (AMO) equipped with an ultra-low penetrating air (ULPA) filter to trap aerosolized particles.

Please use only polystyrene tubes on all analyzers including BD Falcon tubes cat# 352008. CSTs are performed every week on all the instruments to grant optimal performance.

• LSR FORTESSA: Lasers: 405nm, 488nm, 561nm, 633nm. Up to 13 colors detected. LsrFortessa Filter Configuration (pdf).
• CantoII: Lasers: 405nm, 488nm, 633nm. Up to 8 colors detected. CantoII Filter Configuration (pdf).
• FACSCaliburs-3: Lasers: 488nm, 633nm. Up to 4 colors detected. FacsCalibur (pdf).

•  BD X-20: Lasers: 355nm, 405nm, 488nm, 561nm, 633nm. Up to 18 colors detected. BD X-20 Filter Configuration (pdf).

Additional filters are available for the optimal detection of different fluorophores. Please ask the FACS staff if you need to change the default filter configuration of the instrument.

Analysis workstations

Two workstations in the office nearby room 8518 are equipped with FlowJo, BD FACS Diva and FCAP Array analysis software and are available at no charge.

FlowJo site license

The FACS core has acquired a site license for FlowJo. FlowJo is billed/renewed annually. The annual license fee is currently $213 but is pro-rated if the license is less than a year. If you have any questions about FlowJo please contact Erica Lantelme at elantelme@wustl.edu.

At your appointment you must bring to the FACS lab:

Your sample(s): Cells in sorting buffer

Use the buffer that is best for your cells: PBS (no Ca/Mg), PBS/1-2%BCS/FCS, MACS buffer (PBS/2%FBS/1mM EDTA for sticky cells). Cell concentration: About 10-15millions/ml. Minimum volume 200ul. Bring a few mls of additional sorting buffer in case we need to dilute the sample. IMPORTANT: Filter the sample just before sorting throughout a 40 um mesh to avoid clogs. You can also coat collecting tubes with complete medium or FBS/BCS overnight to optimize cell recovery/survival. When sorting few cells for RNA extraction you might consider to sort directly in lysing buffer (i.e. RLT buffer for the Qiagen RNeasy Plus Micro Kit).

Controls

• Negative control (unstained, un-transfected, isotype control.)
• Single stained controls if you have more than one color for setting up the proper compensation.
• FMO controls (if your experiment requires this control).

Optimal concentration for controls: 1×10⁶ cells/ml and a 0.5ml volume.

Collection: Tubes or plates.

• TUBES: 15 ml, 5 ml or 1.5 ml tubes with Collection Buffer. Any liquid you need for your experiment is fine: media, PBS with or w/o serum, lysis buffer. To improve cell survival after sorting it is better to have proteins in the buffer (Serum).
• PLATES: 96-well plates, 384-well plates

For more information on how to prepare your cells and controls email facs@pathology.wustl.edu.

Flow cytometry video tutorials

Fluorescence tutorials (ThermoFisher Scientific)

Introduction to Flow Cytometry (BD Biosciences)

Multicolor Staining Guide (BioLegend)

Spectra viewers

Fluorescence SpectraViewer (ThermoFisher Scientific)

Fluorescence Spectrum Viewer (BD Biosciences)

Fluorescence Spectra Analyzer (BioLegend)