Flow Cytometry & Fluorescence Activated Cell Sorting Core

Director: Marina Cella, MD
Lab Manager: Erica Lantelme, PhD

Phone: 314-362-3562
Email: facs@pathology.wustl.edu

Location: BJCIH Building, Room 8513


Overview

The Flow Cytometry Core provides investigators with instrumentation and support for cell sorting as well as acquisition and analysis of flow cytometry data.

  • High Speed Cell Sorting
  • Assistance with experimental design
  • Instruction and training on the instruments
  • Consultations on sample preparation and data analysis

Access

Service available to: All entities, including for-profit organizations.

Please note that each user needs to have an individual account. First time users: please follow this link to register. When you log in fill in all required information and request access to instruments. Please allow one business day for the activation of your account. Contact the FACS Staff if you have any questions or problems with the new account (facs@pathology.wustl.edu)


Services and pricing

High-speed cell sorting (Staff – operated, AriaII-1)

Appointments with the Staff are available Monday-Friday from 10am-5pm.

Requests for cell sorting can be made using the on-line schedule or by contacting the FACS Staff by email (facs@pathology.wustl.edu). The on-line 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).

High-speed cell sorting (Self– operated, AriaII-2)

Trained operators will be able to reserve time directly on the AriaII-2 schedule. Training consists of 2 sessions in which modalities of sorting and instrument set ups are illustrated (see instructions and training section). People are encouraged to attend the 2 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.

High-speed cell sorting (Staff or self- operated, AriaII-3)

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 (facs@pathology.wustl.edu).

Nozzles available to each sorter:

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

Measuring cell metabolic activities

The flow cytometry core has 2 Seahorse machines (XF-96 and XF-24) to measure ECAR and OCR of cells. For additional information and training please contact Marina Cella (mcella@wustl.edu).

Instructions and training on the instruments/software

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

The on-line schedule is accessible only to trained users. To obtain training, please contact the FACS Staff (facs@pathology.wustl.edu). Once trained, you will be issued a username and password to access the on-line 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:

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

FEES

Instrument fees are based on the following hourly rates billed in 15 min increments for sorters and in 1 min 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.

Instrument Fee ($/hr)
BD FACSAriaII-1/AriaII-3 (BSL2+) Cell Sorters $85.00
BD FACSAriaII-2/AriaII-3 Cell Sorters (Self- Operated) $60.00
BD LsrII $40.00
BD LsrFortessa $35.00
BD CantoII $35.00
BD FACSCaliburs 1 and 3 $20.00

 

The fee for SeaHorse usage is $35.31 per hour. A discount is applied to DRC members.

FACSAriaII-1 cancellations

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

Equipment

BD FACSAriaII cell sorters

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. Click here for details.
  • BD FACSAriaII-2: available for operation by trained users. Lasers: 405nm, 488nm, 633nm. Up to 10 colors detected. Click here for details.
  • 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. Click here for details.

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.

BD FACS analyzers

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

  • LSRII: Lasers: 355nm, 405nm, 488nm, 561nm, 633nm. Up to 15 colors detected. BD High Throughput Sampler (HTS) available for 96 well plates. Click here for details.
  • LSR FORTESSA: Lasers: 405nm, 488nm, 561nm, 633nm. Up to 13 colors detected. Click here for details.
  • CantoII: Lasers: 405nm, 488nm, 633nm. Up to 8 colors detected. Click here for details.
  • FACSCaliburs-1and Calibur-3: Lasers: 488nm, 633nm. Up to 4 colors detected. Click here for details.
  •  BD X-20: Lasers: 355nm, 405nm, 488nm, 561nm, 633nm. Up to 18 colors detected. Click here for details.

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

Seahorse Bioflux analyzers

Two SeaHorse analyzers (XF96 and XF24) are available for operation by trained users.

Useful link: Cell Analysis (Seahorse) Agilent Technologies

Software

Analysis workstations

Two workstations in the office nearby room 8518 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. The cost for the license is $250 per billing year (Oct 1st- Sept 30th). The first license is offered at the discounted price of $100. These fees represent a significant discount over individual purchases. If you have any questions about FlowJo please contact Erica Lantelme.


Technical information

Cell sorting: Information for your appointment

At your appointment you have to bring to the FACS lab:

Your sample(s): cells in sorting buffer

Use the buffer that is better 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 useful links


Detailed contact information

Emails

Director: Marina Cella, MD (mcella@wustl.edu)

Lab Manager/Cell Sorting Operator: Erica Lantelme, PhD (elantelme@wustl.edu)

Cell Sorting Operator: Dorjan Brinja (dbrinja@wustl.edu)

General Lab E-mail: facs@pathology.wustl.edu

Mailing/Shipping address

Flow Cytometry Facility
Department of Pathology and Immunology
Washington University School of Medicine
425 South Euclid, Box 8118
Saint Louis, MO 63110
314-362-3562

Location: BJCIH Building, Room 8513, 8518