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Flow Cytometry Facility

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Flow Cytometry Facility

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Flow Cytometry Facility

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Core Resources

Please review the following resources for the Flow Cytometry Facility to learn about our core operations:

Quick Links
Facility Access
Hours of Operation
  • Regular hours of operation for the Flow Cytometry Facility are:
Monday Closed
Tuesday 9:00 AM – 5:00 PM
Wednesday 9:00 AM – 5:00 PM
Thursday * 9:00 AM – 5:00 PM
Friday Closed
Saturday Closed
Sunday Closed
  • Operator instrument set-up and shutdown occurs between 9:00 AM–10:00 AM and 4:30 PM–5:00 PM, respectively.
  • *Note. Only the BD LSRFortessa X-20 is available on Thursdays.
After Hours / Off-Days
  • The Flow Cytometry Facility currently does not permit the use of its instruments after hours or on off-days.
Training Requirements
Mandatory Safety Training

All users must complete the following safety training before working in the Flow Cytometry Facility:

User Equipment Training
  • User training for the BD LSRFortessa X-20 Cell Analyzer is in-person with the guidance of the Core Manager, a certified equipment operator.
  • The duration of the training is approximately 2 hours.
  • Graduate students, postdocs, research associates, staff, and principal investigators can be trained to use the equipment on their own. Undergraduate students can also be trained; however, Core Manager supervision is required when using the equipment.
  • The cost of training is $100/person and typically a maximum of 3 people can be trained at one time.
Core Policies / Protocols

If you are new user interested in the services offered by the Flow Cytometry Facility, please contact the Core Manager to schedule an initial assessment meeting to learn more about how the facility can help with your research.

Scheduling Equipment Usage
  • To schedule an appointment to use the equipment in the Flow Cytometry Facility, please submit a request through our contact form
Cell Analyzer Booking:
  • The BD LSRFortessa X-20 Cell Analyzer should be booked a minimum of 1 week in advance of its use; however, it may be booked within a week depending on equipment availability.
Cell Analyzer Experimental Information Form
Cell Sorter Booking:
  • The BD FACSAria Fusion™ Cell Sorter must be booked a minimum of 2 weeks in advance of its use.
Cell Sorter Experimental Information Form
Late Arrival
  • Users that are more than 15 minutes late for their scheduled appointment will be charged for the full hour of equipment usage.
Cancellation/No-Show
  • Users that are unable to attend their scheduled appointment at the Flow Cytometry Facility will be charged unless they provide at least 24 hours cancellation notice to the Core Manager.
  • No-shows will be charged the full amount of the booking.

The Flow Cytometry Facility at the University of Windsor has a BSL2 permit; no BSL3 samples are allowed in the facility.

Flow Cytometry Facility Biosafety
  • All requests for analyzing and sorting samples must be accompanied by the Biosafety Form, signed by the principal investigator (PI).
Biosafety Containment Level 1/2 Acknowledgement Form
  • BSL2 samples must be fixed before they are analyzed on the cytometer; these samples include: all infectious, human, and non-human primate cells, as well as cells manipulated with viral agents (e.g., EBV, vaccinia, HTLV, lentivirus, nanoparticle, etc.). If the in vivo model is considered BSL2, then your sample is a BSL2 sample.
  • Primary human samples used for sorting need to have the screening results and be negative for: Hepatitis B, C, HIV, Mycobacterium tuberculosis, Neisseria meningitides, Chlamydia psittacci, Coxiella burnetii, HTLV-1,2, LVMV, vesicular stomatitis virus infections.
  • Samples manipulated with viral agents: EBV, vaccinia, HTLV, lentivirus, nanoparticle, etc. must be clearly described in the Biosafety form. These samples need to be washed at least 3 times and passed through a 70 micron nylon filter just prior to being brought to the Flow Cytometry Facility facility.
  • Gloves should be worn by all users at all times.
  • No food or drink is permitted.
  • During sign-up procedure users must notify the Core Manager about the biosafety level of the material to be analyzed or sorted.
  • No radiolabeled samples are permitted on any instrument.
  • All biological waste must be removed from the facility for disposal in their home laboratory.
Flow Cytometry Facility Data Management:
  • Data collected at the Flow Cytometry Facility is considered temporary storage during experimentation and will not be backed up. Users must save a copy of their raw data to a personal drive and delete any remaining files off the instrument computer at the end of each experiment to maintain storage space for ongoing facility experiments.
  • The Flow Cytometry Facility is not responsible for any lost data. All users should ensure that they have proper plans for data storage, backup, and security prior to using the facility.
Ethanol-Fixation of Samples
  1. Place 1 x 10^6 cells into a polypropylene tube and centrifuge at 1000 rpm for 5 min.
  2. Resuspend the pellet with 1 mL of PBS (1x) and centrifuge 1000 rpm for 5 min.
  3. Remove the supernatant without disturbing the pellet and add (dropwise) 1 mL of -20 C 70% EtOH to the cell pellet while vortexing.
  4. Keep cells on ice for 2 hrs or at -20 C until the day of the staining (cells can be stored for several weeks at -20 C).
  5. On the day of staining, centrifuge the samples 1000 rpm for 5 min, remove the supernatant.
  6. Resuspend the pellet in 1 mL of PBS (1x), centrifuge again, remove the supernatant.
  7. Resuspend the pellet in 0.5 mL of PBS (1x) + 2 mM EDTA and proceed to the staining protocol.
DNA Staining with PI

*Note. The facility does not run samples with PI anymore, please check the DAPI staining protocol below.

Cell Viability with DAPI

Staining of Live Cells for Viability Analysis by Flow Cytometry

  1. Obtain a single cell suspension.
  2. Resuspend cells in BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 1× Dulbecco’s Phosphate Buffered Saline (DPBS) containing 0.05-0.2 μg/mL DAPI.
    • The optimal concentration of DAPI for viability analysis may vary by cell type. We recommend titrating the reagent for your cell type of interest in early experiments.
    • Additionally, apoptotic cells may stain with variable amounts of DAPI. We recommend co-staining with BD Pharmingen™ FITC Annexin V (Cat. No. 556419) if further analysis of apoptotic cells is desired.
  3. Incubate 5 minutes at room temperature. No wash is necessary prior to analysis.
  4. Proceed to analysis by flow cytometry.

Link: BD Biosciences Website

DNA Staining with DAPI

Staining of Fixed Cells for DNA Content Analysis by Flow Cytometry

  1. Obtain a single cell suspension.
  2. Treat cells on ice for 30 minutes with 70-80% ice-cold ethanol.
    • Ethanol fixation typically provides the most resolved histograms. However, this reagent has also been successfully used for DNA content analysis with the Transcription Factor Buffer Set (Cat. No. 562574/ 562725) or BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Phosflow™ Perm Buffer III (Cat. No. 558050) protocol.
  3. Wash cells once with BD Pharmingen™ Stain Buffer (FBS).
  4. Dilute DAPI solution to 0.5-1 μg/mL in Stain Buffer (FBS) or 1× DPBS immediately prior to use.
  5. Stain cells for 5-15 minutes at a cell density of 1 – 2 x 10^6 cells/mL. No further wash is necessary prior to analysis.
    • The optimal cell density and concentration of DAPI for DNA content analysis may vary by cell type. Assay conditions should be optimized in early experiments for best results.
  6. Proceed to analysis by flow cytometry.

Link: BD Biosciences Website

Buffers for Cell Sorting and Analysis
  • 1x PBS (Ca2+/Mg2+ free)0.5 – 2 mM EDTA
  • 1-2% FCS (only for sorting)
  • 0.2 µm filtered, store at 4 °C
  • Or if pH-stability is crucial
  • 1x PBS (Ca2+/Mg2+ free)
  • 1 mM EDTA
  • 25 mM HEPES pH 7.0
  • 1% FCS (heat-inactivated) or BSA
  • 0.2 µm filtered, store at 4 °C
Cell Surface Antibody Staining
Intracellular Antibody Staining
  • Add 25 uL of PI solution (250 ug/ml) to 0.5 ml of PBS (1x) + 2 mM EDTA containing 1 x 10^6 cells of less, keep the samples at least 20 min on ice and covered. Use a 5 ml Falcon BD Flow tube.
Important Forms
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