Flow Cytometry

An Introduction

The Flow Cytometry facility offers a full complement of leading-edge instrumentation from cell-sorting and analysis to the very latest in imaging flow technology. With our specialty in image-enabled flow cytometry and cell sorting, we offer the ability to harness the combined power of imaging, traditional flow cytometry, spectral detection, and machine learning to drive discovery. We get to know our clients and their research question; then, we provide the necessary tools and expertise to assist them throughout every stage of their research from experimental design and training to data analysis and reporting.

Michael Parson wokring on Image Stream

Quick Navigation

Services

The Flow Cytometry facility provides services at two different locations at the Lunenfeld-Tanenbaum Research Institute:
600 University Avenue, Room 980
and
25 Orde Street, Level 4 Room 421.

1. Cell Sorting

We support a variety of full-service cell sorting applications using either conventional or spectral detection for single cell or bulk sorts. Our BD FACSDiscover S8 allows for the inclusion of six additional imaging channels complexed with spectral detection to provide novel cell sorting parameters. For clients wishing to perform their own sorts, we provide comprehensive cell sort training on our Sony MA900 cell sorter(refer to the Equipment section for instrument configurations).

For information on how to prepare samples for cell sorting, refer to our sample preparation guidelines shortform, longform, BD pre sort buffer and please note that cell-sort requests must be accompanied by a biosafety form prior to booking.

Annie Sorting Cells

2. Cell Analysis

Our facilities have a full suite of conventional and image-based flow cytometry analyzers with high-throughput capability. Please refer to the Equipment section for specific instrument configurations.

3. Consultation/Training

For researchers who wish to perform their own flow experiments, we provide comprehensive consultation services that include in-depth theory review, experimental design recommendations, hands-on instrument training with follow-up troubleshooting, data analysis and reporting assistance.

4. Available software with workstations

  • Amnis AI: Amnis AI incorporates advancements in machine learning, including computer-aided hand-tagging, clustering with object map plots, generation of a novel experimental model using deep learning CNN (convolutional neural network) algorithms, and confusion matrix tables with accuracy analytics.
  • IDEAS 6.4: IDEAS 6.4 with ML module is a machine learning enabled analysis software for our Amnis ImageStream available at the facility.
  • IDEAS 6.2: IDEAS 6.2 is the basic Image analysis software for our Amnis ImageStream (contact flow core for copy).
  • FluoroFinder: An experimental design tool integrating panel design tools with spectra viewers, antibody search and a fluorescent dye database. Our facility has registered many instruments and their configurations with FluoroFinder to help get the most out of this tool.
  • ModFit: DNA modeling software with automated analysis and cell tracking capability
  • FlowJo: FlowJo is a comprehensive flow cytometry analysis tool. Clients can acquire a license by following this link.
  • Kaluza: Kaluza is a basic flow cytometry analysis tool. This software is free to use within the LTRI network through a 10-seat network based licensing system. Instructions for downloading and connecting Kaluza to the network.

    • Equipment

    1. Cell Sorters

    BD FacsDiscover S8 (Image-enabled spectral cell sorter):

    Configuration

    Location 25 Orde St, Room L421
    Excitation 349 nm, 405 nm, 488 nm, 561 nm, 637 nm
    Detectors full spectrum – 78 APD fluorescence
    LL+ Scatter Detectors violet laser - axial light loss, SSC
    blue laser (image) – FSC, SSC & Axial light loss
    Image Detectors 488 nm excitation - fluorescence (PMT FL1 LP505:534/46 - FL2 LP570:600/60 – FL3 LP675: 788/225)
    Imaging Features center of mass X/Y, correlation, delta center of mass, diffusivity, eccentricity, max intensity, moment long/short, radial moment, sixe, total intensity
    Nozzles 85 mm, 100 mm, 130 mm
    Sort 4-way, indexed, plate
    BD Facs Discover S8
    Beckman Coulter MoFlo Astrios EQ cell sorter

    Configuration

    Location 25 Orde St, Room L421
    Excitation 355 nm, 405 nm, 488 nm, 561 nm, 642 nm
    Detectors 21 (355 nm/3, 405 nm/3, 488 nm/7, 561 nm/4, 642 nm/4)
    Nozzles 70 mm, 100 mm,130 mm
    Sort 6-way, indexed, mixed mode, plate
    Beckman Coulter MoFlo Astrios
    Sony MA900 Cell Sorter

    Configuration

    Location 25 Orde St, Room L421
    Excitation 405 nm, 488 nm, 561 nm, 638 nm
    Detectors 12 (co-linear 488 nm & 561 nm/5 + co- linear 405 nm & 638 nm/7)
    Nozzles 70 mm, 100 mm,130 mm
    Sort 6-way, indexed, mixed mode, plate
    Sony MA900

    2. Cell Analyzers:

    Amnis ImageStream MarkII (Imaging Flow Cytometer)

    Configuration

    Loacation 25 Orde St, Room L421
    Excitation 405 nm, 488 nm, 561 nm, 592 nm, 638 nm
    Imaging Channels 12 channels (brightfield, darkfield + 10 fluorescent)
    Features Upgraded camera (high gain), Upgraded 488 nm laser (400 mW), 96-well plate autosampler, DEAS v6.4 - machine learning enabled for enhanced image analysis, Amnis AI stand alone deep learning analysis software available
    Image Stream
    BD Fortessa X-20 (HTS)

    Configuration

    Location 600 University Ave, Room 980
    Excitation 355 nm, 405 nm, 488 nm, 561 nm, 640 nm
    Colours 18 (355 nm/2 405 nm/6, 488 nm/2, 561 nm/5, 640/3)
    Features High-throughput sampler
    Fortessa X20
    BD Fortessa (HTS)

    Location 600 University Ave, Room 980
    Excitation 405 nm, 488 nm, 561 nm, 640 nm
    Colours 14 (405 nm/5, 488 nm/2, 561 nm/4, 640/3)
    Features High-throughput sampler
    Fortessa
    ThermoFisher Attune NxT(HTS)

    Configuration

    Location 600 University Ave, Room 980
    Excitation 405 nm, 488 nm, 561 nm, 637 nm
    Colours 14 (405 nm/6, 488 nm/2, 561 nm/3, 640/3)
    Features Acoustic focusing, CytKick Max autosampler (96-, 384-well plates + 24 x 1.5 ml micro-tube rack with cooling
    Attune
    Beckman Coulter Gallios

    Configuration

    Location 25 Orde St, Room L421
    Excitation 405 nm, 488 nm, 561 nm, 640 nm
    Colours 10 (co-linear 488 & 561 nm/5, 405 nm/2, 638 nm/3)
    Features 32 tube carousel

    Pricing

    Pricing is for academic users only. Commercial clients should contact Michael Parsons (parsons@lunenfeld.ca) for pricing.
    Instrument Internal Pricing ($/hr) External Pricing ($/hr)
    Assisted Unassisted Assisted Unassisted
    BD S8 cell sorter $100.00 $75.00 $115.00 $85.00
    MoFlo ASTRIOS cell sorter $90.00 n/a $100.00 n/a
    Sony MA900 cell sorter $90.00 $60.00 $100.00 $65.00
    Amnis ImageStream analyzer $90.00 $65.00 $95.00 $70.00
    Thermo Attune NXT analyzer $85.00 $45.00 $90.00 $50.00
    Beckman Gallios analyzer $85.00 $45.00 $90.00 $50.00
    BD Fortessa X-20 analyzer $85.00 $45.00 $90.00 $50.00
    BD Fortessa analyzer $85.00 $45.00 $90.00 $50.00
    Training and Theory $90.00 n/a $99.00 n/a
    Data Analysis $90.00 $0.00 (1) $99.00 $0.00
    MA900 cell sorter chip $50.00 $50.00 ea. $55.00 $55.00 ea.

    (1) There is no charge for unassisted use of computer workstations and data analysis software.

    Instrument Internal Pricing ($/hr) External Pricing ($/hr)
    Assisted Unassisted Assisted Unassisted
    BD S8 cell sorter $100.00 $75.00 $115.00 $85.00
    MoFlo ASTRIOS cell sorter $90.00 n/a $100.00 n/a
    Sony MA900 cell sorter $90.00 $60.00 $100.00 $65.00
    Amnis ImageStream analyzer $90.00 $65.00 $95.00 $70.00
    Thermo Attune NXT analyzer $85.00 $45.00 $90.00 $50.00
    Beckman Gallios analyzer $85.00 $45.00 $90.00 $50.00
    BD Fortessa X-20 analyzer $85.00 $45.00 $90.00 $50.00
    BD Fortessa analyzer $85.00 $45.00 $90.00 $50.00
    Training and Theory $90.00 n/a $99.00 n/a
    Data Analysis $90.00 $0.00 (1) $99.00 $0.00
    MA900 cell sorter chip $50.00 $50.00 ea. $55.00 $55.00 ea.

    (1) There is no charge for unassisted use of computer workstations and data analysis software.

    Guides

    Training Resources

  • 1. Flow Post-Its from the Whitehead Institute for Biomedical Research
  • 2. Open Flow Cytometry Videos from the Whitehead Institute
  • 3. For Purchase – Comprehensive training program from Work-Flow
  • 4. BD Biosciences Introduction to Flow Cytometry
  • 5. Guidelines for the use of flow cytometry and cell sorting
  • 6. Paul Robinson flow course (comprehensive)
  • 7. Paul Robinson Imaging Course

    • Software/User Guides

    1. Attune NXT
  • a. user guide
  • b. quick guide
  • c. small particle guide
  • d. fluorophore selection guide
  • e. ModFit users guide for Atune
    • 2. Kaluza data analysis
  • a. Kaluza network License set up article
  • b. Kaluza learning center
    • 3. ModFit LT
    4. FlowJo
  • a. How to acquire a license for FlowJo
  • b. CellView Lens Plugin
  • c. FlowJo university
    • 5. BD FACS Discover S8
  • a. S8 users guide
  • b. S8 Quick reference guide
  • c. Fluidics Start up daily / extended start up
  • d. Shutdown daily/long term
  • e. Targeted workflow examples
  • i. GFP basic sort
  • ii. 6-colour
  • iii. GFP imaging
  • iv. 5 colour PBMC sort

  • f. Exporting and converting CVW files for analysis in FlowJo
  • g. Image feature descriptions
  • h. Analyzing data using FlowJo –CellView Lens Plugin

    • 6. Fortessa
  • a. Using DIVA
  • b. HTS operation
  • c. Fortessa 2 configuration
  • d. Fortessa X-20 configuration
    • 7. Sony MA900
  • a. Training guide
  • b. Fluorochrome guide
  • c. Nozzle selection
  • d. Start up
  • e. Sort modes
  • f. Tube sort
  • g. Plate sort
  • h. Useful hints

    • Useful links

    1. Links to spectrum viewers
  • a. BD spectrum viewer user guide
  • b. Thermo Fisher
  • c. Biolegend
  • d. Fluorofinder
    • 2. FPbase Fluorescence spectra viewer
    3. Panel design tool ThermoFisher
    4. BD Panel design tools
    5. FluoroFinder tools
    6. Sample preparation – short form (doc 1), long term (doc 2)
    7. Steps for successful flow by Ryan Duggan
    8. How to use BD Brilliant stain buffer
    9. Become a member of the International Society for the Advancement of Cytometry ISAC to access Cytometry topics of interest.

    Publishing Standards guide for Flow Cytometry
    MIFlowCyt

    Policies

    Hours of Operation

  • Facility staff are available between the hours of 9:00 am and 5:00 pm Monday to Friday. Staff will endeavor to accommodate clients outside of these hours upon special request however, additional charges may apply.
  • Assisted Cell sorting appointments start at 10:00 am with the last appointment beginning no later than 3:00 pm unless special provisions have been made for assistance outside of normal working hours.
  • Access to the facility after hours is restricted to approved Lunenfeld-Tanenbaum Research Institute (LTRI) personnel with appropriate training.  After hour access for users external to the LTRI requires the presence of facility staff due to restricted building access and LTRI safety policies.

    • Getting Started

  • All new users must review these policies and complete a registration form prior to commencing work.
  • Quick overview of facility Guide
  • Human patient samples may require further REB approval (contact parsons@lunenfeld.ca)
  • Trainees are requested to review information in our getting started document. Training consists of a two-hour theory review followed by hands-on instrument orientation regardless of previous training from other facilities. Unassisted sample acquisition is granted once competency has been demonstrated. Requests for training can be made by contacting core facility staff (parsons@lunenfeld.ca).

    • Biosafety

    A biosafety questionnaire must be completed by all new clients and approved by facility staff before work commences and all users must review these guidelines:

  • General safety information can be found online under LTRI Safety.
  • Gloves and lab coats are mandatory.
  • No radiolabeled samples are permitted on any instrument.
  • LTRI biosafety requires that procedures for handling biological specimens conform with practices outlined in Laboratory Biosafety Guidelines (Public Health Agency of Canada-PHAC and enforced by the human Pathogens and Toxins act).
  • Users of our facility can sort live class II biohazardous samples – RG2 (e.g. all virally infected cells or cell lines of human origin). We cannot sort or analyze specimens meeting RG3/4 criteria. For clarification of RG2 human pathogens, see Human Pathogens and Toxins Act.
  • Specimens meeting RG2 typically can be fixed using a suitable method prior to analysis to reduce the sample to RG1 classification. Prior to commencing work, the facility may request a test involving the use of a cell-viability dye to ensure the efficacy of fixation. Under special circumstances, provisions for analyzing live RG2 specimens may be granted with prior approval.
  • Handling of RG2 samples involve aerosol mitigation strategies such as working in approved biosafety cabinets, centrifugation in rotors with safety caps, and vortexing with sample tubes capped.
  • Clients are asked to remove any biological waste for disposal in their home laboratory. As a shared facility, this is both a precaution for staff using the facility as well as a requirement for maintaining a sterile sorting environment.

    • Billing and Reservations

  • All instrument bookings are handled through the Resource scheduler.
  • Instrument usage charges pertain to a combination of time booked on the Resource scheduler and actual instrument logs.
  • Reservations requiring operator assistance can be made by viewing the facilities resource scheduler and contacting the operator to make a reservation.
  • A 48 hr. cancelation policy is in effect for assisted services (not including weekends and holidays). Contact the operator directly for cancelations. No shows or late arrivals may be charged for the full reservation. Requests for operator assistance outside of normal hours are available as a special request (additional charges up to 1.5X normal rate may apply).
  • Clients will have access to the facility’s calendars to view availability and make reservations. Reservations can also be made through request to staff.
  • Qualified clientele will receive logins for instruments on which they have been trained.
  • Please note that actual instrument usage is logged for each client and reconciled with calendar reservations. Charges will apply to the higher of the two.  Instrument use without reservation may be charged at 1.5X normal rate at the discretion of the facility.
  • Reservations for unassisted instrument use may be canceled prior to 24 hours ahead of the booking (not including weekends and holidays) and it is the responsibility of the client to make the cancelation. No shows will be charged for the full reservation.
  • Reservations can be switched to a different client by contacting core staff if the reservation is past the 24 hour notice period. Please note that charges will be made to the reservation holder’s account, so it is important to ensure the reservation is changed to the correct client.
  • Usage is billed every quarter. Please note that the facility only guarantees that instruments are working within design specifications and makes no guarantees of positive experimental outcomes.

    • Client Responsibilities

  • Clients are responsible for following start-up and shut down procedures (quick reference guides are available next to the instrument).
  • Clients are required to perform a complete shutdown of the instrument if they are the last user of the day or if there is two or more hours to the next booking.

    • Data Management

  • Data storage is the sole responsibility of the client and clients are urged to transfer data to their own storage devices as soon as possible. Data will be removed regularly from the instrument workstation since it adversely affects instrument performance.
  • Data storage on the dedicated analysis workstations will be removed when necessary (and without notice), so please use access to the LTRI network drives available on the analysis workstations to save your data or store directly to external storage devices.
  • Clients may log on to the instrument’s work station to transfer data. However, please note that off-line instrument analysis should be done using the available analysis workstations or client’s personal computer since the instrument log cannot distinguish between actual instrument usage and analysis and charges may be applied accordingly.

    • Acknowledgement

    Preferred acknowledgment for the Network Biology Collaborative Centre Flow Cytometry Facility:
    The authors wish to thank [staff name] of the Network Biology Collaborative Centre Flow Cytometry Facility (RRID: SCR_025384) at the Lunenfeld-Tanenbaum Research Institute for [service]. The facility is supported by the Canada Foundation for Innovation and the Ontario Government.

    Click here for our publication policy and preferred acknowledgement for other NBCC Facilities.

    NBCC

    Network Biology Collaborative Centre

    Sinai Health, Lunenfeld-Tanenbaum Research Institute
    600 University Avenue, Room 970
    Toronto, Ontario Canada M5G 1X5

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