FAQ
What are the advantages of sorting by Flow Cytometry compared to other isolation techniques (i.e. magnetic bead separation, panning, etc.)?

Fluorescence Activated Cell Sorting (FACS) provides a method for purifying a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
FACS offers several advantages compared to other cell isolation techniques. It provides the possibility to separate populations of cells based on their fluorescence intensity level, offers better separation of populations by using multidimensional sort parameters (positive or negative selection based on expression of several different markers at the same time) and can separate cells based upon internal 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 various kinds of collection devices while eliminating dead cells.

How much will I be charged for using the facility resorces?

Invoices are sent every 3 months with a minimum charge of 100 CHF per trimester to cover administration costs. This money contributes to the running costs of all the machines.

Invoices ar send by the accounting department of the University of Lausanne. For any inquiries concerning the invoices please contact

pricelist
What type of cells/particles can be sorted?

Any type of cells or particles, in the range of 0.1µm to 35µm, and which can be kept in a single cell suspension, can theoretically be sorted. Cells that tend to clump, agglutinate or settle out of suspension easily can be problematic during sorting, as this will cause clogging of the nozzle and disturbance of proper droplet formation. Primary Human samples or samples infected with BSL2 pathogens require special handling and biosafety precautions. Many BSL2 classified cells or particles can be sorted on our site by following BSL2 practices, however as cell sorting is an aerosol producing technique, some BSL2 classified samples must be handled as BSL3 which is currently not possible on our site.

For more information please refer to the Biosafety section of this website.

What effect will the sorting process have on my cell's viability?

The effects of fluorescence activated cell sorting on viable cells is dependent on factors such as cell type, condition of the cells prior to being sorted, pressure/jet velocity at which cells are sorted and the buffer the sample is to be sorted into.
Although cells of many types have been sorted and thereafter grown in culture, it is advisable to remember that there are no guarantees that every cell type will hold up under every manipulation. Seidl et al [18] investigated effects of magnetic separation (using the MACS system from Milrenyi Biotec) or droplet sorting on a BD FACStarPLUS on membrane integrity (propidium iodide exclusion), microviscosity, membrane potential, and Annexin-V staining in breast cancer cells and normal skin fibroblasts. Both separation techniques, as well as ancillary preparative steps such as enzymatic cell dissociation, affected membrane physiology, but the authors felt that neither magnetic separation nor droplet sorting was clearly preferable for cell preparation[19].

The free advice is this: Don't sort when you don't have to. If you are looking for rare events, and/or want to isolate large numbers of cells, take advantage of every available bulk separation technique as an alternative to sorting and/or to pre-enrich samples for the population(s) you want. These days, immunomagnetic separation, for which Dynal, Immunicon, and Miltenyi, amongst others, provide a variety of reagents and apparatus, offers a wide range of options.

What concentration and medium should my cells be in and what should I bring for my sorted sample?

Again this depends on your cell type. Some cells tend to clump at higher densities. In general cells should be at a concentration between 10-15 x 10E6 cells/ml in a nutrient-supplemented, protein-containing medium. Often simple PBS containing 3% FCS (fetal calf serum) buffered at pH 7.2 is sufficient, and doesn't result in excessively cell death. If using Ca2+ or Mg2+ dependent antibodies or fusion proteins a Ca2+/Mg2+ containing salt solution (such as modified Hanks' Balanced Salt Solution (HBSS) Invitrogen Catalog Number 14025076) might be used. For very sensitive cell types which require more complex media a modified RPMI-1640 (with L-glutamine) (Sigma, Product number R8755-10X1L) which is commercially prepared without biotin, riboflavin, phenol red, and bicarbonate might be an option.
Generally for all staining buffers biotin should be avoided since it will interfere with avidin second-step reagents. Riboflavin and phenol red should be eliminated because they can increase apparent cell autofluorescence. The pH is buffered at 7.2 with 10 mM HEPES, and the protein source s hould be heat inactivated 3% newborn or fetal calf serum or bovine serum albumin.

For 1 x 10E6 sorted cells of each population bring one 12 x 75mm Falcon polypropylene tube per sorted population with approx. 1ml of support media which contains 20% FCS (or some other source of protein such as BSA) and antibiotic (pen/strep or gentamicin); or (2-way sort only) for 5 x 106 sorted cells of each population bring one 15ml Conical tube per population with approx. 3ml of support media which contains 20% FCS (or some other source of protein such as BSA) and antibiotic (pen/strep or gentamicin). For more information please refer to the Sample Preparation for FACSAria Cell Sorter information sheet available from the download section of this website.

What type of information do I need to provide about my sample and why is this necessary?

In order to have a successful sort and to properly configure the instrument, the following information is required at the time of reservation, or at least 48 hours before the sorting experiment.
In the download section of this website you can find a registration form for cell sorting in which you need to supply all the following information.

  • What type of cells do you want to sort? Please supply information about the species, the tissue origin, if it is a primary cell or cell line etc. This is important for establishing instrument settings and pressures.
  • What is the approximate size of your cells? This is very important in selecting the nozzle size to be used.
  • What has been done to the cells, e.g. have they been stimulated, drug treated, transfected or infected? This is important in choosing nozzle size and jet velocity.
  • Which fluorescent stains have been used to label the cells? This is important in selecting lasers and optical filters and in setting compensation for spectral overlap. It is important to discuss these issues prior to setting up your experiment to ensure the instrument has the ability to excite, detect and compensate your fluorochrome combination.
  • What will be done with the sorted sample? e.g. short term culture, long term culture, PCR, functional assays, etc, this will help determine the sterility conditions as well as jet velocity.
  • Into what kind of collection device do you wish to sort? e.g. Eppendorf tubes, 12 x 75mm tubes, 15ml Conical Tubes, 96 well plates, 24 well plates, 6 well plates, microscope slides, this is important for choosing the right collection device holder, jet velocity and instrument setup.
  • How many populations do you wish to sort simultaneously? (1-4 populations). Note: Only one population can be sorted for plate and slide sorting.
What is yield and recovery and how many cells do I need to bring?

Yield is defined as the number of cells that have physically been sorted divided by the actual number of cells within your gate of interest. An increase of sample rate (number cells/second analyzed) and the presence of cell aggregates and debris can increase the losses and ultimately reduce your yield.
The two key factors influencing the sorting yield are:

 

electronic aborts – these appear if cells arrive at the laser interrogation point too close to one another; in that case the system cannot precisely determine to which event (cell) the measured signal should be allocated, therefore it aborts both events.

 

sort conflicts – these appear if an actual positive cell fails the sort criteria e.g. if the desired event is too close to a potentially undesired event, the machine will abort (not sort) the desired event to ensure purity. There are other modes of sorting that favor recovery over purity, so if total cell numbers are more important than purity, this can be accommodated.

 

Recovery is defined as the number of cells you can harvest from your tube after the sort, divided by the number of cells that have actually been sorted. This number varies between cell types and is due to cells sticking to the tube, getting lost in centrifugation steps and dying during sorting. In high speed sorting mode cells are accelerated to a speed of 97 km/h within a split second, pass through a 70um orifice, receive 50 to 100Volts, pass an electric field of up to 6,000Volts and hit the surface of the collection tube or plate reducing their speed to 0km/h within microseconds. So getting sorted is usually not that much of a vacation for your cells.

 

So how many cells you will finally need to bring, depends on how many cells you need to recover for your further studies, the frequency of the target population of interest, the % sorting yield (generally 75-95%) and finally the % recovery. It is always best to calculate the number of input cells based on the worst-case scenario of a 70% yield rate and 70% recovery so you will be sure to have enough cells to reach the desired number.
For example, if the target cell population you are interested in sorting is 10% of your unsorted cells (total events above threshold) and you need to recover 1 x10E6 target cells, you would need
~ 2.0 x 10E7 cells as an approximate starting cell number.

 

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