Protocols

Sample Preparation for Cell Sorting (MoFlo-XDP)

For sorting, a minimum of 10 x 106 cells is usually required; the optimal concentration is between 3-5 x 106 cells/ml. Cells ready for sorting should be resuspended and then collected in the media they are "happiest" in, which includes either commercially available FACS buffers, PBS, or cell culture media such as RPMI or DMEM. Realize that the presence of phenol-red in the culture media may affect the background florescence slightly; however, in general this is not a problem.

In the case of adherent or inherently sticky cells, it will help to add 1mM EDTA to the sorting buffer. Adding EDTA should prevent the formation of cell clumps, which will be aborted by the machine and not sorted. Also, addition of a buffering agent like HEPES (pH 7.0) to the sorting and collection buffers will help buffer the pH of the solution and aid in cell viability as the cells exit the sorter. Bring the collection buffer of choice as well as extra sorting buffer in case the cells need to be diluted.