How long will it take to sort my cells?

       The shortest possible sort time to collect a defined number of cells, given a starting sample with different levels (percent positive) of cells to be sorted, is shown below.  The calculation is based on a fairly typical analysis rate of 2500 cells/second:

 

Cells to Collect

STARTING (SOURCE) Percent POSITIVE
0.1% 1.0% 5.0% 10.0% 50.0%
103 6.7 min 40 sec 8 sec 4 sec 1 sec
104 1.1 hr 6.7 min 1.3 min 40 sec 8 sec
105 11.1 hr 1.1 hr 13.3 min 6.7 min 1.3 min
106 4.6 days 11.1 hr 2.2 hr 1.1 hr 13.3 min
107 1.5 months 4.6 days 22.2 hr 11.1 hr 2.2 hr
108 1.3 years 1.5 months 9.3 days 4.6 days 22.2 hr

 

What can I do if the sort times above are unreasonable?

        Your only option is to enrich your starting sample by some means (i.e. remove negative cells or concentrate positive cells by antibody mediated panning, lysis or adherence to beads).  One can also sort, in the enrich mode on the flow cytometer, a smaller number of cells and put these back into culture for expansion which will (hopefully) result in a population with a higher percentage of positives.

 

What do I need to bring for a cell sorting run?

        Your cell suspension should be at a concentration of between 2-5 x 106/ml and free of cell aggregates and large debris particles (they will clog the instrument and end your sort).  Thus, it is advisable to filter your cells through a 30-40µ nylon mesh to remove aggregates and then do anything you can to prevent re-aggregation (keep them on ice, remove Ca++ and Mg++ from buffer, add EDTA, etc.).  The cells should be suspended in a medium which does not stress the cells (i.e. at a minimum, PBS with 1% BSA is fine).  Tissue culture media is acceptable but it should be without a pH indicator dye as many of these will increase the background fluorescence and thus reduce your signal to noise separation.

        For the collection of sorted cells you will need to bring collection media augmented to suit your cells.  This may range from simple PBS up to enriched tissue culture media and, if the cells are particularly sensitive to lowered serum concentrations, should have double the normal amount of serum.  The additional serum will compensate for the dilution of the PBS sheath fluid which makes up most of the actual droplet that is sorted.

 

Sorting options.

        A recirculating water bath is available to keep the sample and the sorted cells at a constant temperature.  If temperature control is necessary be sure to inform the laboratory personnel when you schedule your sort time so that the water bath can be set up and stabilized before you arrive to sort.

        An Automated Cell Deposition Unit (ACDU) is also available for sorting a defined number of cells into the wells of microtiter plates or various sized tissue culture plates. The user may select  1 or more cells/well and the number can be varied within a single plate.  Again, be sure to let the laboratory personnel know ahead of time if you want to use this option.