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Protocol

Please note that PBS is used for the sheath fluid.  It is the user’s responsibility to provide appropriate buffer if PBS proves to be unsuitable.

A. Cell Preparation for cell sorting

  • It is imperative to provide necessary controls for your sort experiment. If you do not have the proper controls, or fail to adjust cell concentration based on an accurate count, we will highlight there is no guarantee regarding the outcome of the sort, during your session.
  • We maintain the fluidics system of the instrument in a clean condition. It is essential to prepare controls and samples under sterile conditions and to use sterile and endotoxin free buffer/tubes to prevent contamination of the fluidics system with LPS and potential microorganisms.
  • It is essential to filter your samples through a cell strainer or a BD Falcon 35 µm filter cap immediately before sorting.
  • Please prepare more cells if you can. As the sort yield (the total number of cells sorted in the collection tube) is influenced by threshold rate, flow rate, sample quality, target cell frequency etc., it is sensible to assume that the sort yield will be 50% of the theoretical yield. For low frequency/rare cells populations it is recommended to enrich your sample for the population of interest. This will result in a much quicker sort and a far better purity, viability and yield
  • We advise you to include a live/dead marker in your staining panel (please try to exclude PI).  This is particularly important when you are analysing low frequency/rare cell populations since dead cells have a tendency to bind to the staining antibodies non-specifically, leading to staining artefacts.
  • To set up the machine, you need to provide 1x 105 non-stained (negative control) cells in 500 ul volume. Provide single stained cells, FMO control or comp beads to set up compensation and target gates in a minimum volume of 200 ul. If you are unsure of what controls to provide please consult us.
  • Labeled cells should be diluted in a phenol free media (such as RMI or HBSS) or calcium-magnesium free PBS supplemented with 1-2% fetal calf serum (FCS) at a cell concentration of 15-20 x 106 per ml for sorting at 45-70 psi or 5-10 x 106 cells per ml for sorting at 20 psi.   Please bring additional media in case it is necessary to dilute your samples further.
  • If your cells are adherent or particularly sticky or have previously been frozen, to prevent cells from clumping during the sort please include 2-5 mM EDTA in all steps of the sample preparation. DNase can be used at 10 ug/ml if EDTA proves to be unsuitable for your sample type.

B. Collection tubes

  • We recommend using polypropylene (PP) tubes for both samples and collected tubes. Cells are less likely to adhere to these tubes than to polystyrene (PS) tubes.
  • To prevent sorted cells sticking to the walls of the collection tubes, these tubes should be coated with FCS or any serum of your choice. We recommend including 100-500 ul neat FCS or your serum of choice into the collection tubes. This will help cell recovery and increase cell viability. The amount of serum in the collection tubes should be adjusted if you require a defined serum concentration at the end of the sort.

It is recommended to use eppendorf or 5 ml tubes if you expect to obtain small number of cells and 15 ml tubes if you expect to obtain large number of cells.