Cell Analytics Facility

The Cell Analytics Facility is a core service of the University of Fribourg. It provides access to the instrumentation, technical expertise and training to perform experiments requiring the technique of Flow Cytometry. We provide a cell sorting service as well as advice on experimental design, sample preparation, data analysis and support on flow cytometry techniques and instrumentation. We are here to help you get knowledge about flow cytometry, from experimental design to data analysis.

Currently the facility is equipped with a FACSCalibur, a MACSQuant cell analyzer, a BD LSRFortessa as well as a FACSAria Fusion cell sorter.

Our services and facility are available to UniFR research staff, but also to researchers from other universities or companies. Please contact us if you have any questions, ideas or particular needs.

  • Services

    What we do:

    • Maintenance, quality check and troubleshooting on all instruments
    • Provide training and guidance in the use of those instruments
    • Provide all relevant information on our website
    • Provide a booking system
    • Spread knowledge on flow cytometry through teaching, workshops and hands-on training
    • Offer a service of cell sorting using BD FACSARIA Fusion instrument
    • Provide help on request for experimental panel design, sample preparation and data analysis

     

    What we don’t do:

    • Experiments for you
    • Data analysis for you
    • Install software or anything on your own computer

     

  • Flow Cytometry and Experiment Information

    Basics

     

    What is flow cytometry?

    Flow Cytometry is a technique allowing the analysis of single eukaryotic cells or particles from different sizes, such as viruses, bacteria, beads, yeast etc. in suspension, based on their size, complexity and fluorescence. Briefly, this technique analyzes single events passing through the different lasers beams, detecting light scattering properties and fluorescence emitted by the cells.

     

     

     

    What is a Fluorescence Activated Cell Sorter (FACS)?

    FACS is a subtype of Flow Cytometry allowing the active sorting of several cell populations from a hetero­geneous mixture based on the cell specific light scattering and fluorescence properties.

    Using high voltage plate deflectors, a FACS instrument can separate individual cells from the bulk population and collect them in a separate tube. Samples labeled with all conventional fluorochromes and fluorescent proteins can be processed.

     

     

     

    Experimental design

     

    How to design correctly your experiment

    Some basic rules should be considered when preparing to build a panel and choosing a combination of fluorochromes:

    • Know your hypothesis and your sample

    • Know your instrument (lasers + filters)
      • It will give you the limitation and possibilities of fluorochromes, predict potential problems

    • Know your antigen
      • Use only monoclonal antibodies for FC
      • Choose the appropriate antibody clone, check for expression level in the literature.

    • Know your fluorochromes
      • The relative brightness (use published staining index to get an idea)
      • The type (is it a tandem dye)
      • The stability (tandem dyes can degrade, some dyes don’t support fixation, some others need special buffers when combined,...)

    • Build your panel
      • You should use bright fluorochromes for less-expressed antigen and vice versa.
      • Chose fluorochromes with minimal spectral overlap (more compensation will give more data spread), often better on different lasers.
      • Use online tools such as spectra viewers, Fuorofinder or Chromocyte to help you.
      • Put non-critical markers on less good channels.
      • Don’t forget to add viability dye and dump channel if you can! This will help you reduce background. Put them in “bad” channels. Here some tips on fluorochromes combinations
    • Optimize your reagents
      • When your panel is ready, titrate each antibody!
      • Use Fc blocking if you are working in presence of antigen-presenting cells (avoid unspecific binding).

     

     How to prepare your sample

    A sample that is prepared in the best possible way will always give you better results.  Follow these points to get good data:

    • Your cells should be in single cell suspension (use PBS-based solution with 1-3mM EDTA and 1-3% protein)
    • Protect your samples from the light (fluorochromes are light-sensitive)
    • Keep your samples on ice (to avoid changes of your condition)
    • Best concentration around 5-10 million cells/ml (maximum 20 million/ml)
    • Filter your sample through a 40 micron cell strainer just before

     What controls to have

    Proper controls are both important for sorting and for non-sorting flow cytometry, as they allow for correct settings and gating of your cells of interest. Without controls, you don’t know what you are analyzing

    • Unstained cells: ALWAYS (which had the same treatment as your cells of interest) to set the negative signal and check for auto-fluorescence.
    • Single stainings: ALWAYS if more then 1 color (tubes containing only one of the used antibodies, all separated). It’s required to compensate. We recommend using compensation beads!
    • FMO: GOOD to check data spread due to other fluorochromes. It’s a tube where you put all antibodies except the one of interest. Mandatory to put correctly the gate for rare events and low separated population.
    • Isotypes: BAD only to check Fc block… most of the time useless
    • Reference control: GOOD to compare between experiment (frozen PBMCs, beads)
    • Positive: GOOD to prove that you experiment works and to check for run to run variation (can be your reference control).

     

    Analysis


    You need to use your own computer for analysis, as we do not provide any computer with analysis software. If you need help for the analysis, do not hesitate to contact us – we are happy to assist you.

    Our staff members mostly use FlowJo software for analysis. You will find some references to standard flow cytometry analysis software under the Links section.

    If you are a member of the Faculty of Science and Medicine, you can apply for a FlowJO license (limited number, shared licenses). If you are interested, please send an email. We will come back to you with information on how to get and use the license. 

    For UNIFR members, you can download the FlowJo software and install it on your UNIFR computers following these links:

    PC users

    MAC users

     

  • How to get started

    New users

    You need to register by filling out this form with all your information and send it to us by e-mail. We will contact you within 48h to fix a date for the practical training (1h) on the instrument of interest. After completion of the training, you will be granted access to the booking system of the instrument you have been trained to use.

     

    Facility users

    If you need access to another instrument, please send us your request by e-mail. We will contact you within 48h to fix a date for the theoretical (1h) and practical training (1h) on the instrument of interest. After completion of the training, you will be granted access to the booking system of the requested instrument.

     

    Cell Sorting Service

    If you want to use our Cell Sorting instrument for your experiment, please check the instrument availability in the  online booking calendar, fill out the Cell Sorting Form accordingly and send it to us by e-mail. We will contact you within 48h to fix a meeting to discuss your project and your need for the instrument. After that, our staff will confirm your booking and an operator will perform the sort. If you are not a member of the University of Fribourg, please contact our staff first.

     

  • Conditions of use

    General rules

    As a user, you and your supervisor (PI) agree and are bound to:

    • Complete the training specific to each instrument you intend to use
    • Access the instrument using your personal login only (which is granted upon completion of the training)
    • Treat the material and instruments carefully at all times
    • Filter every sample before running it on any instrument, using your own filtering material
    • Follow the procedures for startup, cleaning and shutdown as described during practical training
    • Report any irregularities to the facility staff immediately
    • Book via the official facility instrument booking system before using a cytometer
    • Be responsible for shutting down the instrument you have been using, if you are the last user on that day
    • Please note:  there is no guarantee on device performance or experiment outcome.
    • Any damage to the instrument due to bad usage (not following the rules, policies and SOPs) will be billed to the user responsible for the damage.

     

    Booking and cancellation

    • Booking is mandatory and must be done through the OPEN Iris online booking system. Only trained users may book and use instruments.
    • Please play fair and match actual instrument usage with your booking time (fees are calculated based on bookings).
    • If the booking doesn’t match the actual usage several times, the user will be charged 200.- for bad usage.

    • The analyzers can be booked 7day/7 between 8.00 and 23.00, for a duration of up to 4 hours. Bookings can be cancelled until 30 min before beginning. You can shorten an ongoing booking if you need less time than expected, but you cannot delete it.
    • The cell sorter can be booked every weekday between 9.00 and 17.00. A cell sorting session has a minimum duration of 1 hour and can only be booked by a staff member (the calendar is available online on Open IRIS). Sorts can be cancelled up to the day before without charge. Cancellations made less than 24 hours before the start of your booking will be fully charged.

     

    Data management

    • Please bring a clean and virus-free USB key to transfer “fcs” generated data files on your own computer. On certain instruments, a transfer to the file server is possible as well.
    • Every two months, all data will be deleted from the instruments without notice. Users are responsible to save and backup their data elsewhere.
    • All data that can be observed in the Cell Analytics Facility is strictly private and may not be used or copied for personal experimentation.

     

    Fees

    • Instrument uses are billed automatically based on the entries in the booking system.
    • Invoices and payment requests will be issued every 3 months.
    • Every year, the fees will be adapted depending on the number of operating hours of each instrument (the more people use it, the less expensive it will be). Currently, the usage fees are:

     

     

    Members of UniFR

     

    External academics
    (prices incl. operator)

    External non-academics
    (prices incl. operator and data analysis)

    FACSCalibur Cell Analyzer 10 CHF/hour NA NA
    MACSQuant Cell Analyzer 35 CHF/hour 50 CHF/hour 100 CHF/hour
    FACSARIA Fusion 150 CHF/hour 200 CHF/hour 300 CHF/hour

     

     

  • Booking System

    Before getting access to the booking system, you must complete a theoretical (1h) and practical training (1h) on the specific instrument (see How to get started). Booking information for the Cell Sorter can be found in the Flow Cytometer Cell Sorter section.

     

    If you are a new user, you need to register by filling out this form and sending it to us by e-mail.

    If you are a facility user and need access to another instrument, please send us your request by e-mail

     

    All instrument booking is done using  Open IRIS with the following procedure.

  • Flow Cytometer Analyzer

     MACSQuant

     

    The MACSQuant from Milltenyi is a flow cytometer analyzer equipped with 3 lasers: a 488nm blue laser, a 638nm red diode laser and a 405nm violet laser. MACSQuant allows the detection of 8 simultaneous fluorochromes and runs MACSQuantify software.

     

    Optical configuration of the MACSQuant analyzer

    Instructions for standard tasks on the Miltenyi MACSQuant

    SOPs

     

       


    Below you can find a schematic representation of the MACSQuant's optics.

     

     

    BD LSRFortessa

     

    The Fortessa from BD Bioscience is a flow cytometer analyzer equipped with 3 lasers: a 488nm blue laser, a 640nm red diode laser and a 405nm violet laser.

    The key features of this system include a fluidics system that uses hydrodynamic focusing, a patented optics system, integrated software for acquisition and analysis, and options to enhance the workflow.

    Our BD LSRFortessa instrument allows the detection of 11 simultaneous fluorochromes (13 parameters) and is running under BD FACSDIVA 6.0 software.

    You can find here the optical configuration of the instrument with some of the fluorochromes that you can detect. 

     

    Optical configuration

    SOPs

     

     

     

    FACSCalibur

     

    The FACSCalibur from BD Biosciences is a flow cytometer analyzer equipped with 2 lasers: a 488nm blue laser and a 640nm red diode laser. FACSCalibur allows the detection of 4 simultaneous fluorochromes and runs CellQuest software.

     

    Optical configuration of the FACSCalibur analyzer

     

     

     

    Below you can find a schematic representation of the FACSCalibur's optics.

     

     

     

  • Flow Cytometer Cell Sorter

    FACSAria Fusion

     

    Please note that, because of its complexity and sensitivity, this cell sorter can only be booked and operated by our staff.

     

    The operator will process your samples on the instrument. You are required to stay until the right parameters are set for the sorting of your cells, and to come to pick your cells after sorting for further processing.

     

       

     

     

    Specifications

    • The FACSAria Fusion is a 3-laser cell sorter that can measure at the same time up to 14 fluorescence parameters and 2 lights scatter parameters. In addition to being placed in a hood, this instrument has an aerosol containment system. 
    • On the FACSAria Fusion a maximum of 4 independent cell populations can be sorted in parallel from the same starting sample tube.
    • BSL-2 samples can be sorted.
    • Various collection vessels can be used: 15 ml, 5 ml, 2 ml and 1.5 ml tubes, slides and multi-wells plates.

     

    Optical configuration of the FACSAria cell sorter

     

     

    Application

    • Our FACSAria Fusion is mainly dedicated to the isolation of eukaryotic nucleated cells. Yeast, bacteria, nanomaterial or any other special applications must be discussed with the facility staff first.
    • Sorting is performed inside a biosafety cabinet under BSL2 conditions.
    • Any population of cells inside a more complex heterogeneous-population can be isolated, as long as the cells of interest can be identified in the parental population by light scattering and fluorescence (for example: GFP-infected cells or antigen-marker populations using fluoro-labeled antibodies).
    • The FACS technique offers some advantages compared to other isolation techniques. It can separate populations of cells based on their fluorescence intensity level and can separate cells based upon intra­cellular staining (DNA, cytokine expression, eGFP etc.). Usually FACS offers a higher purity, provides a higher recovery of cells of interest and can sort a specified number of 1-4 different cell populations within one sample into diverse collection devices (tubes, plates), while eliminating dead cells.
    • Sorting does affect the cells! As they are in suspension for a long time, cells will be stressed. Mem­brane physiology can be affected by the charging, the flow will induce shear stress, and both cell meta­bolism, signalling and gene expression can be affected. Think twice and do not sort cells if you do not have to!

     

     

    Sample preparation

    • Your cells should be prepared under the best possible conditions to preserve viability (gentle dissociation, suitable medium, 4°C). 
The happier the cells are, the better yields you will get.
    • Samples should be single-cell suspensions of about 10-20 millions per ml in Ca/Mg++ free HBSS or PBS buffer supplemented with 1-3% protein (either BSA or FCS) and 1-3 mM EDTA to minimize clustering. 

    • The cell concentration of samples for a plate sort (single cell cloning) should not exceed 1.5 million cells/ml and should not go below 0.5 million cells/ml.
    • Count cells right before sorting, not before staining procedure, which often results in considerable cell loss. Always start staining with more cells than needed for the sort (e.g. twice the number). 

    • Please re-suspend your cells carefully before coming to the sort and filter them through a 40 micron cell strainer on a 5ml FACS tube just before coming to the sort.
    • Your cell samples should be kept on ice and protected from light.
    • Be aware that rare population will require a longer sorting time just because of the frequency of the detected vents (for example if you want 100’000 cells that are 0.1% of your sample it will take 1 hour).
    • For rare populations, whenever possible, please pre-enrich them using MACS columns to reduce sorting times and recover cells in better shape.
    • To preserve cell size and morphology after fixation protocols, put cells back into PBS/FCS or similar for running on the machines. 

    • We strongly recommend using viability dyes to exclude dead cells.

     

     

    Things to bring with you for a sort

    • Your samples
    • A sample of unstained cells (mandatory for the instrument set-up)
    • Single color controls (mandatory for compensation, if more then 1 color) on beads or on cells
    • Extra-buffer to dilute your sample if needed
    • Sterile medium to collect your sorted cells, around 1-2 ml per tube
    • USB key to transfer the data

     

     

    Access and Booking

    Cell sorting is a time-consuming technique that requires careful planning. Since the operator will perform the sorting, you need to pre-book your experiment at least one week in advance.

    Please be aware that some users need to book the sorters for a whole day. You are therefore advised to plan and book your experiments well in advance and to discuss your experimental protocol with us to ensure that sorting is doable.

    For booking, please first check the cell sorter availability on the  Open IRIS calendar, send the completed Sorting Form to us by e-mail. We will confirm your booking usually within 48h, and we will evaluate the time you need at the instrument. If it is your first booking, we will briefly meet to discuss your experiment protocol. There is a minimum of 1 hour booking slot for sorting. Don’t hesitate to contact us when planning your experiments, especially if you do it for the first time!

     

     

  • Contact

    If you want to become a new user of the facility or if you want to follow the training for another instrument, please see the "How to get started" section.

    For any question regarding flow cytometry please contact us via e-mail.

     

    Cell Analytics Facility Staff

    Facility Coordinator Curzio Rüegg
    Facility Manager Sarah Cattin

     

    Contact 

        +41 26 300 85 79

       cellanalytics@unifr.ch

     

    Location

     

    PER17

    Chemin du Musée 18

    CH–1700 Fribourg

     

       

     

  • Links