Fluorescence Activated Cell Sorting

1. Cell Preparation: The goal is to prepare a sample containing as many single cells as possible. Doublets or higher order clumps will either be detected or thrown out by the cytometer, or will confuse the instrument and result in sort mistakes (resulting in reduced purity). To avoid this, cells should be filtered through a 30-100 μm nylon mesh, depending on cell size, at each step to continually remove cell aggregates. Cell aggregation occurs during centrifugation washes and can lead to tremendous losses of cells due to mega-clumps. This can be minimized by using use round bottom tubes. Do not use conical or tapered bottom tubes. Use a relatively large diameter tube e.g. 17mm. Polypropylene is the best plastic to use with cells – do not use polystyrene. Centrifuge cells only as fast as necessary to pellet. Do not leave the cells in the pellet for a significant time it is best to be at the centrifuge when it stops spinning and immediately remove tubes, decant supernatant (never suck off fluid using a pipette) and resuspend cells (hint: break pellet up before adding any additional fluid/media).