Protocols

Sample preparation

Please note that sterile 1X PBS is used for the sheath fluid. It is the user’s responsibility to check that this buffer is appropriate for their cell type.

• It is imperative to provide necessary controls for your sort experiment. If you do not have the proper controls, fail to adjust cell concentration based on an accurate count, or decide to proceed with the sort against the facility manager’s advice, we will not be held responsible for the outcome of the sort.
• 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/or microorganisms. If you are uncertain of whether your procedure for sample preparation is appropriate, seek guidance from the Facility Manager.
• Keeping cells in a single cell suspension is critical for successful sorting. It is crucial to filter your cells through a 35 µm nylon mesh before sorting.
• Please prepare more cells if you can. The sort yield (the total number of cells sorted in the collection tube) is influenced by threshold rate (cells/second), flow rate, sample quality, and target cell frequency. Thus, 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 previously enrich your sample for the population of interest (for example via magnetic beads isolation/enrichment). This will result in shorter sort time, and enhanced 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 in a non-specific manner, leading to staining artefacts.
• To set up the machine, you need to provide 1 x 10⁵ non-stained (negative control) cells in 500 µl volume. Provide single stained cells, FMO control or comp beads to set up compensation and target gates in a minimum volume of 200 µl. If you are unsure of what controls to provide please consult the Facility Manager.
• If your cells are adherent or particularly sticky or have previously been frozen, you should include 2-5 mM EDTA in all steps of the sample preparation to prevent cells from clumping during the sort. For samples with a high percentage of dead cells, DNase must be added at 10 µg/ml to the sample.
• Labelled cells should be diluted in a phenol-free media (such as RPMI or HBSS) or calcium-magnesium free PBS supplemented with 1-2% foetal calf serum (FCS). Please bring additional media in case it is necessary to dilute your samples further.

Collection tubes

We recommend using polypropylene (PP) tubes for both samples and collection 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 should be coated with neat FCS or any serum of your choice. We recommend including 100-500 µl FCS or your serum of choice into the collection tubes to help cell recovery and increase cell viability.

You could also provide your media of choice supplemented with desired serum concentration. It is recommended to use Eppendorf or 5ml tubes if you expect a low yield of sorted cells, and 15ml tubes if you expect a high yield.