Flow Cytometry Core Sort Preparation :: Nationwide Children's Hospital

Optimizing Cell Sorting

The single most important factor affecting the quality of a cell sort is proper sample preparation. If you have a less than optimal sample you’ll probably get a less than optimal sort. Junk going in generally means junk coming out.

There are numerous factors that impact a successful cell sort. Because there are many different types of cells used and different objectives involved in the sort there is no single method of preparation that can cover all situations. That being said, what follows are some of the issues that each investigator should consider in order to make their particular sort as successful as possible.
 

Cell Size and Morphology

Investigators can sort anything from small, spherical resting lymphocytes to large, aged cells with fragile processes. Because of these differences in cell size and architecture it is important that each cell population be matched to a particular nozzle size on the sorter. The calibration of the sorter is matched to each nozzle size in order to create an optimal stream velocity and droplet breakoff. A drifting breakoff can disrupt the optimized settings of the sorter, especially the droplet delay, resulting in the disruption of side streams in which the desired sample misses the collection tube. This disruption, as a result of changing the droplet breakoff, can have a deleterious effect on sort purity.  In a worse case scenario a mismatched cell/nozzle combination can result in the clogging of the nozzle.

Morphology is an even trickier factor due to the variation between cell types. Generally, as cells deviate away from the ideal spheroidal shape the more suseceptible they become to shear forces from the sort stream that can end up damaging said cells. Generally, the more a cell deviates from the ideal sphere the larger the nozzle required in order to do an effective sort. For this reason it is very important that the investigator relay information regarding cell morphology in addition to cell size to the sort staff prior to a sort.
 

Sample Preparation

As mentioned above the single most important factor in a successful sort is proper sample preparation. It doesn’t matter how well the sorter has been calibrated and optimized if the sample itself is inferior.
 

Single Cell Suspensions

In order for the sorter to work properly cells must be in a single cell suspension throughout the duration of the sort. This is an especially important factor when working with adherent “sticky” cell lines or tissue homogenates. Proper sample preparation will go a long way toward optimizing your sort.
 

Sample Concentration

Cells must be at the proper concentration in order for the sorter to work as designed. Samples that are too concentrated will suffer a lower recovery due to the fact that the sorters inability to distinguish between separate events will cause it to abort an event in order to maintain purity. On the other hand, cells that are too dilute will have a much longer processing time. Having said this there is no one universal optimal sample concentration, as cell size, morphology, tip size and sort rate all have an impact in this arena. A good general starting concentration for large, cultured and or sticky cells is 1 million cells/ml whereas blood cells can be put at 5 to 6 million cells/ml. With these concentrations as a starting point one can then go on to determine the best concentration empirically.
 

Sort Buffer

This is a very important factor in achieving an optimized  sort. A proper buffer will help to keep the cells in a “happy” state as well as help to maintain a single cell suspension. Depending on cell type, various additives and or changes to the basic buffer recipe can help toward optimizing the quality of your cell prep. Please take a look at these Sort Buffer recipes to see if they may help in optimizing your sample.

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