Fluorescence-activated cell sorting is a powerful tool for basic and clinical research because individual cells can be separated from a heterogeneous sample and used for downstream analysis or therapeutic applications. A fluorescent activated cell sorter works in a similar way as a flow cytometer. A single-cell suspension of fluorescently labeled cells pass through a fluidic system, and lasers excite the fluorescent molecules, which causes a change in the charge of the droplet containing the cell. This shift in charge is used to divert each droplet into a collection tube so relatively pure cell populations can be collected. Cell sorting can be done by any researcher, but many scientists work with contract research organizations that have expertise optimizing protocols for different yields or levels of purity.

Consider these five fundamentals of cell sorting as you plan or coordinate your next research project.

1. How many cells in and out?

Consider how many cells you want to include in your pre-sort sample because this will determine how long your sort will take and how many cells you will collect. More cells in the starting sample can lead to greater yields but can take longer to sort.

2. How rare are the cells you are sorting?

If you are planning to sort T cells from peripheral blood, then you have a relatively abundant fraction of cells to work with. If you are trying to sort a rare subset of T cells or another rare population like dendritic cells, you will need to start with a larger pre-sort sample so you can end up with a reasonable number of sorted cells.

3. What are you doing with your cells?

The conditions for cell sorting are also determined by what you are doing with your sorted sample. If you are using sorted cells for small scale in vitro experiments, you are likely to need fewer total sorted cells than if you are using them for animal studies. If you are using cells for clinical studies in humans, not only will you need more cells, but cells will have to be sorted under specific conditions to meet safety and regulatory requirements.

4. Are you using any pre-sort purification protocols?

Cell sorting can be done more rapidly and result in greater sample purity if a pre-sort purification step is used. This typically includes a magnetic-bead based separation step, which can selectively enrich your sample or exclude cell populations not being sorted. Additional purification steps can affect cell viability as well, so consider this factor as well for your experimental outcomes.

5. What nozzle size is being used on the cell sorter?

Cell sorters can be fitted with nozzles of different diameters (such as 70, 85, or 100 µ) to optimize sorting protocols. The nozzle size dictates the size of the cell droplet that is sorted, and a larger diameter nozzle results in a slower sort but can be gentler on cells.

These five factors can help you plan a cell sorting experiment or guide your discussion with a cell sorting expert. Always consider running a pilot sorting experiment under different conditions to optimize your protocol and work out any technical kinks.