User Guide to your First Sort

Please read the FlowCore General Use Policy document.

Bookings should contain as much information as possible, and the appropriate nozzle size selected.  Incorrect nozzle selection means any changes will come out of your booking time.

As a general rule, primary blood or lymph tissue should be booked with a 70um nozzle.  All other tissue types and cultured cell lines should be booked with 100um nozzle.

If you are unsure, please check with FlowCore staff.

FlowCore is a PC2 facility and should be treated as such.  You must do a safety induction on your first visit.  Bags, jackets, etc are to be left in the office. Similarly, the office is not a PC2 space so there are to be no samples in the office at any time.

Samples must be contained in 5ml capped Polypropylene tubes, carried in a sealable plastic container or esky (as per Monash University PC2 guidelines).

You must know what tissue you are bringing in to the facility and inform staff.  If primary human tissue it must be screened.  For human cell lines we must be made aware of any transfections or transductions.  Staff must be made aware of any potential hazards to them or other users.

You must know what surface markers / fluorescent colours you are using.  It is strongly recommended that you titrate your antibodies for maximum resolution.

Please work on having a planned gating strategy and an idea of the populations you are interested in sorting.  This will allow us to provide a better level of assistance.

You must bring appropriate collection tubes and media.  These can be 1.5ml eppendorf, 5ml tubes, 15ml tubes, 50ml tubes, or 96 well plates.  Staff must be notified of any potentially hazardous collection media.

Samples to be prepared ideally at 10 x 106 cells per ml, resuspended in a buffer of your choice, but without PH indicator.  Samples can be prepared at a higher concentration, and diluted by bringing a diluent of your choice with you.

Samples must consist of a cells only control, along with single colour controls for each fluorescent marker, and your sort sample(s).

FlowCore recommends the use of a viability marker for your samples, the most common being Propidium Iododie (PI), or DAPI.

If you are unsure of the capability of the sorters to detect your colours of interest, it is your responsibility to check the website.  If still in doubt check with FlowCore staff.  There are slight differences across the sorters, and some difference in sorters and analysers.  In some cases, combinations suitable for one instrument will not work on another.

Thermofisher Flow Cytometry Protocols Handbook