Fluorescence-activated cell sorting

Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry where a heterogeneous mixture of cell suspension can be sorted into up to four populations of interest based on specific light scattering and fluorescent characteristics.

Cells are sorted one a time into single-cell-containing droplets that are broken off from the stream by a vibrating mechanism.

As droplets flow pass laser beam and light detectors, those with cell of interest that meets the preset criteria are placed with an electrical charge and deflected to different angles before entering designated collection tubes.

FACS can be used for:

  • Establishment of monoclonal transfected cell line by single cell isolation
  • Isolation of particular cell sub-populations of interest (e.g., GFP-positive transfected cells, lineage negative bone marrow cells by surface marker staining)
  • Purification of rare populations such as cancer stem cells

Equipment

Fees

Services are billed hourly unless otherwise stated.

Fees only apply to the actual cell sorting process from when your tube is loaded till when the sorting is done. No fees for pre-sorting consultation, instrument setup (at least an hour for aseptic sort]) laser warm-up and instrument shutdown processes of the sorting.

Item University of Manitoba Industry
First 40 hours  $70 $150
After the first 40 hours  $40 $150
Late fee  $55 $55

Late fees are billed hourly for each hour past the appointment start time. For example, starting two hours late will incur a $110 late charge.

Sample preparation guidelines

Investigators must read and comply with the following guidelines for preparing samples for cell sorting to avoid potential rejection of the sample. Consult the core facility manager if you have any questions or concerns.

Contact the facility manager no later than 24 hours before the scheduled sort to confirm or cancel your appointment.

Review the sort logic and experimental design with the facility manager. Be prepared to supply the following information:

  • preferred date/time of the sort
  • cell type and cell origin
  • approximate cell count in the sample and percentage of the target cell population(s)
  • experimental design and flouro-chrome combination
  • downstream application for the sorted cells
  • sterility requirements

Filter samples before sorting begins to remove any cell aggregates by running the sample through a sterile 40-80μm nylon mesh in a bio-safety cabinet.

Store samples in sterile-capped tubes (12x75mm snap-cap tube or 15ml conical tube) with proper labeling.

Provide collection tubes partially (1/4-1/3) filled with appropriate collection medium.

Select from of the following collection media:

  • Culture media with at least 20% FBS
  • PBS if collecting cells for RNA or DNA
  • Fetal bovine serum only for very fragile cells

Re-suspend your cell sample in appropriate sorting buffer at the proper concentration:

  • 1x PBS (Ca/Mg2+free)
  • 1% FBS or BSA
  • 25mM HEPES pH7.0
  • 1mM EDTA (optional, use it if sample cells are sticky such as DCs, granulocytes, fibroblast, epithelial cells)

Resuspend cells in sorting buffer containing 10 units/ml DNAse during the sorting – this is for cells with high dead cell concentration, to avoid clumping of the cells in the sample by the released DNA.

Concentrate the samples to be sorted. Note that the sorting speed (and thus the length of the time for sorting a particular sample) will be determined by several factors including nozzle size (which depends on your cell size) and your sample concentration and total volume. The larger the cells, the larger the nozzles to be used and thus the slower the sorting speed and the longer the sorting time. The lower your sample concentration and the larger the total volume, the longer sorting time.

It is crucial to prepare your sample to the correct concentration for the best sorting results.

Prepare your sample in a concentration close to the values shown in the table below. You should bring extra sorting buffer in case if your sample is too concentrated.  Minimum concentration of the sample is 10 million cells per ml.

ample concentration (cells/ml
Sorting speed/nozzle size Cell type examples Sample concentration (cells/ml)

high speed/70μm nozzle

splenocytes, lymphocyte, mice bone marrow cells

30-40 million

medium speed/85μm nozzle

cell lines

20 million

low speed/100μm nozzle

DCs, macrophage, granulocytes

10 million

Include all the appropriate controls to set up the instrument for cell sorting. This includes:

  • At least one negative (unstained and/or isotype; bring extra for initial setup if it is your first time sorting the sample) for each cell type/origin.
  • One compensation sample for every fluorochome or dye that you use in your staining. You can use your own cells, or ideally compensation beads, for compensation controls.

Ensure your compensation controls have a distinct sharp clear positive peak that has no overlap with the negative peak. Control tubes should be at 1x106 cells per ml.

You should have a clear idea of the scattering profile and the fluorescent patterns of your target cells before starting the sorting. We recommend performing flow cytometry analysis of your samples first. Generally, the more discrete the target population from the unwanted cells, the better the purity and the yield of the sorting

Please allow for one to several times of test sorting to work out the optimal sorting conditions during the initial trial of sorting new cell samples.

Flow cytometry cell analysis

Flow cytometry is a laser-based technology that allows for simultaneous multi-parametric analysis of physical and/or chemical characteristics of cells at the single-cell level.

Fluorescence-labeled cells are carried to sensing area in sheath fluid and cross laser beam one a time. Information about the properties of cells, including cell size, granularity by scattered light and relative fluorescence intensity, are transmitted to light detectors after passing through a series of optical filters. This information is then converted into digital electronic signals and collected by specialized computer programs for further analysis.

Flow cytometry technique can be used in a wide range of research applications such as:

  • Activation states and oxidative burst
  • Cell viability, apoptosis and proliferation
  • DNA content and cell cycle analysis
  • Gene expression and transfer (e.g., transfection efficiency)
  • Immunophenotyping
  • Intracellular calcium flux
  • Simultaneous analysis of levels of surface and intracellular markers (e.g., intracellular cytokine level)

Equipment

Fees

Sample acquisition by academic trained users

Option 1: Pay-as-you-go

  • Academics: $55/hour
  • Industry: $110/hour

Option 2: Annual one-time capped fee

  • Become our major users by a simple one-time payment of $5,000/year which will give you unlimited access to any analyzers.

Sample acquisition and analysis by core facility supervisor

Fast, reliable and professional work guaranteed. We will work with you along the way to ensure you get quality results that suit your need, whether it is for paper publication or for grant application.

  • Academics: $70/hour
  • Industry: $150/hour

FlowJo site licence program

A FlowJo Portal site license is a user-based license management system for a group or institution. FlowJo Portal provides an improved management interface to site administrators.

Fees

Costs for licences are billed annually.

  University of Manitoba Industry
Per licence  $400 $800

If you buy two licences from us, a third is available at $200 per year.

Fluorescence microscopy

A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances.

What we will do 

Fast, reliable and professional work guaranteed. We will work with you along the way to ensure you get quality results that suit your need, whether it is for paper publication or for grant application.

Investigators are welcome to operate fluorescence microscopy equipment themselves following training. Learn more about training opportunities here.

Equipment

Fees

Sample acquisition by academic trained users

Option 1 

  • Zeiss Confocal, academics: $20/hour
  • Zeiss Confocal, industry: $40/hour 

Option 2:

Annual one-time capped fee. Become one of our major users by a simple one-time payment of $4,000/year which will give you unlimited access to any analyzer.

Sample acquisition and analysis by core facility supervisor

Fast, reliable and professional work guaranteed. We will work with you along the way to ensure you get quality results that suit your need, whether it is for paper publication or for grant application.

  • Zeiss Confocal, academics: $40/hour
  • Zeiss Confocal, industry: $80/hour 

Immunophenotyping

Immunophenotyping is a technique used to study the protein expressed by cells.

What we will do 

Whether it is human PBMCs from peripheral blood or infiltrating immune cells in solid tumour, we can provide you with full service from cell staining, sample acquisition to post-acquisition data analysis.

We offer four panels of bio-markers for human immunophenotyping. If you would like to make modifications on these panels, please contact us.

Panel 1: General Immunophenotyping

BioMarker Fluorochrome Clone
CD3 APCH7 SK7
CD14 FITC M5E2
PDLI (CD274) PE-Cy7 MIH1
CD19 APC HIB19
CD56 PerCP-Cy5.5 B159
CD49d PE 3G8
NKp46 V450 P44-8
LiveDead AMCyan Amine reactive

Panel 2: Naive, effector, memory and regulatory T-cells

BioMarker Fluorochrome Clone
CD3 APCH7 SK7
CD4 V450 RPA-T4
CD8a PE-Cy5.5 RPA-T8
CD25 APC M-A251
CD45RA PE-Cy7 HI30
CD127 FITC HIL-7R-M21
CCR7 (CD197) PE 150503
Dump: CD14/CD19/LiveDead AMCyan Amine reactive

Panel 3: T-cell activation and exhaustion

BioMarker Fluorochrome Clone
CD3 APCH7 SK7
CD4 V450 RPA-T4
CD8a PE-Cy5.5 RPA-T8
CD69 PE-Cy7 FN50
CXCR5 PE MU5UBEE
PD-1 (CD279) APC MIH4
LAG-3 FITC 17B4
Dump: CD14/CD19/LiveDead AMCyan Amine reactive

Panel 4: B-cell activation and regulatory B-cells

BioMarker Fluorochrome Clone
CD19 APC HIB19
CD38 APC-H7 HB7
CD27 PerCP-Cy5.5 M-T271
Ig-kappa FITC TB28-2
Ig-lambda PE 1-155-2
CD69 PE-Cy7 FN50
CD24 BV421 ML5
Dump: CD14/LiveDead AMCyan Amine reactive

What you need to supply

Prepare your cells in single cell suspension and let us take care of the rest.

Special considerations

Users can choose to substitute other markers in the panels if desired, but there will be added fees for the additional staining antibody.

Fees

Services are billed hourly unless otherwise stated.

Item University of Manitoba Industry
Performed by investigator Free Free
Performed by facility $70 $150

We also provide antibodies and reagents at a charge of $20 per panel per sample if you do not want to purchase your own antibodies.

Contact us

Flow Cytometry
413 Apotex Center, 750 McDermot Avenue
University of Manitoba
Winnipeg, MB, R3E 0T5, Canada

204-294-0691
204-789-3921