Overview

Flow cytometry is the science of examining physical and chemical properties of live cells, beads or other biological particles as they pass in a fluid stream through a measuring apparatus. This apparatus, known as a flow cytometer, uses laser excitation and signal detection to measure parameters such as size, shape, DNA content, surface receptors, enzyme activity, membrane permeability and calcium flux. Some flow cytometers, called sorters, are equipped to separate and collect cells or beads of interest.  High-performance flow cytometers and high-speed cell sorters have applications in cancer and HIV research, drug discovery, stem cell and gene therapy. 

The Ottawa Hospital Research Institute Flow Cytometry Facility, located at the General Campus of the Ottawa Hospital, was established in 1998 and now includes three flow cytometers, two of which have sorting capabilities.  We offer the sorting precision required for the isolation of rare cell populations with high purity and yield, up to 20 000 events per second.  As well as sorting, flow cytometers allow the simultaneous analysis of a variety of light scatter and fluorescence parameters on single cells. 

Please follow the Sample Submission Guidelines when using the Flow Cytometry Facility services. 

News and Updates

The FACS facility is excited to announce that another upgrade to the MoFlo has been installed!

In addition to the four new lasers purchased for the MoFlo in 2010, we have added a new violet (405 nm) laser.

This laser optimally excites CFP, as well as other dyes in the blue region of the spectrum, allowing for use of multiple fluorescent proteins in your samples, including FRET analysis between CFP and YFP.

Instrumentation

BeckmanCoulter MoFlo

Location: Ottawa Hospital Sprott Centre for Stem Cell research, 5th floor Critical Care Wing, room 5145, 501 Smyth Road

The BeckmanCoulter MoFlo can be used for multi-parameter experiments and applications requiring high speed sorting and/or sorting of four sub-populations simultaneously.  A designated operator processes the samples.

Configuration

• Jet-in-air system.
• Five excitation lasers:
  
  • Coherent Genesis CX 355nm (UV)
  • Coherente Cube 405nm
  • Coherent Saphire 488nm
  • Coherent Saphire 561nm
  • Coherente Cube 640nm

• Two light scatter channels and 10 fluorescence detectors:
• 3 fluorescence detectors on the 405nm laser path
• 5 fluorescence detectors on the 488nm laser path
• 2 fluorescence detectors on the 561nm  laser path
• 3 fluorescence detectors on the 640nm laser path

 A policy has been implement which states that users of the facilily recognize the potential for contamination of their samples and that use of the MoFlo facility includes this assumed risk. The MoFlo does not generally have a high incident rate with contamination issues; however, as with any sample manipulation, the posbility does exist. In the case of post-storting contamination, it is heavily recommended that the users notify the facility so that proper action can be taken.

Booking Policies

A biosafety information form must be filled out and signed by the PI for each new experiment that will require flow acquisition and sorting on the BeckmanCoulter MoFlo. 

Please send your booking requests to poleynik@ohri.ca.  For each session, the Sample Information Form must be completed and submitted by 9h am on the day of the appointment.  The completed form can be dropped-off at the flow lab (CCW5, W5145), sent via FAX (739-6294) or e-mail (poleynik@ohri.ca).

Services Provided

Flow cytometry

• Immunophenotyping/immunofluorescence by monoclonal antibodies.
• Total/Absolute population counts.
• Analysis of surface and intracellular fluorescent labelling, simultaneously.
• Monitoring gene transfer and expression using fluorescent proteins.
• Simultaneous detection and sorting of GFP and YFP.
• DNA content and cell cycle analysis using PI, Hoechst, BrDU, etc.
• Hoechst 33342 side population.
• Cell viability with exposure to drugs, toxins, etc.
• Apoptosis. 
• Caspase activity.
• Telomere length measurement.
  • Neutrophil functions (phagocytosis and oxidative burst).
  • NK cytolytic activity.
  • MLR / CTL cytotoxicity.

• Caspase activity.
• Mitochondrial function (DiO, JC-1)
• Intracellular Calcium Flux.
• Q-Prep whole blood lysis.

Cell Sorting

• High-speed cell sorting.
• Cell sorting of one to four subpopulations.
• Large particle sorting
• Single cell deposition in multi-well plates.

Training

• Flow cytometry basic training is intended for students, technicians and investigators wanting to strengthen their backround and understanding of flow cytometry.  The 1.5 hour session will provide the participants with the required tools for setting up successful flow cytometry experiments. 

Next sessions:

To be determined.

• Specific instrument training is offered on a as-needed basis, please contact the instrument administrators for further details.

Data Analysis

Different software are available for the analysis of flow cytometry data.  Please contact the instrument administrators for more details.

Summit v. 4.3 (Dako)

• Available for MoFlo users, please contact instrument operator.
• Data acquisition on MoFlo.
• General data analysis.
• Database management.
• Histogram substraction and overlays.
• Up to 10 million event files.

CXP (Beckman Coulter)

• Data acquisition on the Cytomics FC 500.
• General data analysis.

Expo32 (Beckman Coulter)

• Data acquisition on Epics ALTRA.
• General data analysis.
• Easy integration of cytometry graphics and results into text documents, spreadsheets and presentation.
• Powerful overlay tool with three data display modes.

MFI (Eric Martz)

• Freeware available at MFI
• Corrected median fluorescence intensity evaluation.

WinMDI 2.8 (Joe Trotter)

• Freeware available at WinMDI
• General listmode data analysis.
• Improved black & white Colormaps for publication hardcopy
• The Format Print Page allows for easy page composition.

Other OHRI Flow Cytometry Services

 Configuration

• Flow chamber system.
• Three excitation laser: UV
  
  • Argon 488nm
  • HeNe 525nm, 575nm, 610nm, 675nm
• Two light scatter and seven fluorescence detectors

• Flow cell system.
• One excitation laser:
  
  • Argon 488nm
• Two light scatter and five fluorescence detectors

Contacts and Fee Structure

   * An instrument set-up charge applies with each cell sorting experiment.

Links

Instrumentation

• Beckman-Coulter
• BD Canada
• Dako

Reagents

• BD Pharmingen
• Cedarlane
• Chromaprobe
• Dako
• eBioscience
  
• Jackson ImmunoResearch Lab

Safety

• ISAC Biosafety Guidelines

  Spectra

• BD Fluorescence Spectrum Viewer

Societies and Associations

•  Canadian Cytometry Association 
• GLIIFCA

International Society for Analytical Cytometry

 Clinical Cytometry Society

Universities and Institutes

• Perdue University Cytometry
  
• Salk Institute
• University of Chicago
• IRCM