Oregon Stem Cell Center Flow Cytometry Core
Getting Ready for a Sort?
Discuss your cytometry project with the core operator or the director in advance. This will be especially important if you are transfecting disabled pathogens or sorting human cells. Please bring written details on the type of cell to be sorted, cell concentration, markers to be evaluated, and fluorochromes that will be used.The optimal cell concentration will depend on nozzle, pressure, and other factors. These should be discussed with the core operator prior to the day of the sort, though generally a concentration of 10 million per mL is fine with the 70 micron nozzle, 5 million per mL with the 150 micron nozzle.
Samples should be brought in 12x75 polypropylene round-bottom tubes, though, in a pinch, 12x75 polystyrene will also work for the sample tube. Collection tubes must, however, be polypropylene. (We have them if you don’t.)
All sample tubes should be clearly marked to indicate contents. Unless requested otherwise, samples will be named and saved as the tubes are marked.
Live cell sort samples should be put in 1% fetal calf serum or bovine albumin buffer. HEPES can be helpful. The buffer should be phenol red- and biotin-free.
Cells must be filtered, and for cells that like to aggregate, it would be best if they were filtered immediately before going on the sorter. If cell clumping is a problem, addition of EDTA at up to 5mM may help in reducing cation-dependent cell adhesion. If cell clumping is due to released DNA, addition of Dnase II at 10 U/ml can help.
In general, all sorting and analytical experiments should include appropriate negative controls, as well as single color positive controls on all the fluorochromes to be compensated.
If the collected cells are to be cultured or injected into animals, notify the operator when scheduling the sort, so that the instrument can be set up for a sterile sort.
Last, but not least, please provide an account alias and an FAID number.