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Tips for Setting Up a Successful Cell Sorting Experiment

Use the proper cell dissociation method

Using the wrong cell dissociation method can kill your experiment before you even start. Therefore, it is important to consider the type of dissociation method used with respect to the cell type being sorted.

Titrate your antibodies

Excess antibody will bind at low affinity, increase background, and reduce the resolution of your desired population. Titrating antibodies improves the signal-to-noise ratio, reduces nonspecific binding, and increases sensitivity.

Filter and gate your samples

Clogs are a cell sorter’s worst nightmare. They interrupt the sorting process, and interruptions to sorting prolong the time cells are out of ideal incubation chamber conditions. Filtering the sample using a cell strainer just prior to sorting removes cell clumps and helps ensure single-cell suspension.

Use EDTA to avoid clumps

Use a 1–5 mM concentration of EDTA in your sort buffer, if possible, to help break up cell clumps if your cells are sticky and filtering doesn’t solve the problem.

Include serum or protein in your buffers

This will increase the viability of your cells in all phases of the experiment, from straining and washing to sorting. Using 1–3% BSA (preferred) or FBS in your buffers will help keep the cells healthy and viable without making them too sticky. Coating your collection tubes with protein prior to sorting will help reduce the chance that sorted cells will stick to the sides of the tube where they could dry out.

Use a viability dye

The presence of dead cells affects staining and therefore quality of data. Antibodies bind to dead cells indiscriminately. Dead cells have greater autofluorescence and increased nonspecific binding than live cells, which leads to false positives and lower sort purity.

Use compensation controls

No or incorrect compensation for spillover from using single stains prior to sorting can lead to sorting of the incorrect population, thereby reducing the purity of your final sort.

Use FMOs

Florescence minus one are important when building multicolor flow cytometry panels as they will help you determine where to set your gates.

Use the right temperature

Getting the correct temperature for your cell type is important, be it hot or cold.

Know your sorter

Finally, make sure you know everything that you can about the cell sorter you are using for your experiment.


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