10 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.

Adherent cells are typically detached with a combination of trypsin and EDTA. However, trypsin is known for stripping cell surface proteins that are important for fluorescence-activated cell sorting (FACS) (Luckey et al. 2001). If you have to use an enzymatic method, you will likely be better off using a trypsin alternative, as they are better for keeping the surface marker levels unchanged while preserving cell viability. A non-enzymatic dissociation method, such as Gibco Cell Dissociation Buffer, is best for the gentle dissociation of mammalian cells for studies that require intact cell surface proteins (for instance, flow cytometry).

If you use trypsin, you might also want to use a soybean-based trypsin inhibitor to neutralize trypsin activity. The quickest and most common trypsin neutralization method is to add fetal bovine serum (FBS) to the cells. However, unlike soybean-based trypsin inhibitors, FBS reintroduces factors that cause cells to re-adhere to each other, potentially resulting in cell clumping.

If your cell type does not work with either of the methods mentioned above, you can use another nonenzymatic method, such as gentle mechanical dissociation (that is, scraping the plate). Cell scraping causes more physical damage to the cells. But overall, its effect on the cell membrane protein expression or activity is small. This method is particularly useful for cells grown in serum-free media, where you would want to avoid using serum to neutralize trypsin.

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.

Make sure you’re using the right concentration of every reagent. The ideal concentration of antibody is found through calculation of the staining index, which is the ratio of separation between positive and negative populations. The procedure for titrating antibodies requires that an equal number of cells be stained in a serial dilution of antibody while maintaining a constant sample buffer volume. The dilution that represents the best stain index is the dilution to use.

Note that some reagents work better at room temperature. This can be true for antibody staining as well as live/dead staining. Make sure to review the reagent product data sheet for optimal use conditions. You can learn more about titrating your antibody here.

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.

Along with filtration, single-cell gates help ensure that your sort droplet contains only a single cell from the population that you are interested in and not a doublet containing one correct cell and one undesirable cell. You can find sterile cell strainers here.

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. Just be aware that EDTA is known to downregulate some receptors and should not be used when detecting intracellular cytokines or some surface markers that require Ca²⁺ ions, such as integrins. In this situation, use Ca²⁺Mg²⁺-free buffer (for instance, phosphate-buffered saline without magnesium or calcium) or sodium citrate to inhibit clumps.

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. This will increase your sort yield and cellular recovery.

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. So eliminating them is a must. By including a viability dye in your sample, you can gate out dead cells and debris and ensure that only viable cells are being isolated.

It is worth highlighting that for sorting experiments, most viability dyes require serum-free staining. This can be an issue for some cell types, where serum starvation can trigger apoptosis. At the very least it can alter basal cellular metabolism, potentially affecting your experiment (Pirkmajer and Chibalin 2011). So it’s a balance. You can always use more viability dye to make up for the dye taken up by the proteins in solution. Or you can take advantage of a viability dye that can be used with a serum-containing buffer. Use the correct concentration of viability dye and follow the manufacturer’s protocol.

Some dyes, such as 7-AAD, can penetrate cells over time if not washed off, thereby reducing the pool of live cells available to sort. Because of this, viability dyes like 7-AAD and propidium iodide (PI) should really be added immediately before running your sample. Also, adding too much can make compensation more difficult. However, this can be an advantage in that these dyes offer real-time assessment of live/dead populations. Amine dyes that are washed off during the staining process would not be able to stain cells that subsequently die.

In general use DAPI, PI, or 7-AAD for unfixed cells. For fixed cells, use fixable compatible dyes, amine dyes, or Bio-Rad’s VivaFix Cell Viability Assays.

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. For fluorophores that have overlapping emission spectra, you must use single-stained controls. The instrument’s software can calculate the correct spillover values to ensure good data. Compensation should be carried out using the experimental sample where possible. This ensures that the brightness of the markers closely matches that of the sample to be sorted. But if you find yourself in a situation where you don’t have enough sample (for instance, cell number is too low or population too rare), compensation beads stained with your experimental antibody can be used as a substitute.

Use FMOs

Fluorescence minus one (FMO) controls are important when building multicolor flow cytometry panels as they will help you determine where to set your gates. FMO controls are used when trying to identify a true positive from a negative population and when expression levels are low. Fluorescence spread, especially with brighter fluorophores, occurs when acquiring data. It is particularly noticeable after compensation and cross-laser excitation. Careful experimental design and avoidance of channels that have a large amount of spreading will help reduce this influence, but FMO controls are still important.

Use the right temperature

Getting the correct temperature for your cell type is important, be it hot or cold. Keeping cells on ice can slow metabolic processes, reduce gene and protein expression changes, and preserve viability. But make sure your cell type can tolerate low temperatures. Mammalian cells are happiest at 37°C, and some cell types will die quickly if sorted in a 4°C buffer. Cold temperature prevents them from repairing sort-induced damage. So the longer the sort, the more damage that can’t be repaired. If you’re using a mechanistic cell probe, you’ll be better off keeping the cell warm as well. Also, keep in mind that temperature and duration of binding can also affect reagent kinetics. You can always check the literature for sorting protocols for your cell type of interest to see how successful sorts have historically been carried out.

Know your sorter

Finally, make sure you know everything that you can about the cell sorter you are using for your experiment. Understand the excitation and emission wavelengths the FACS instrument can support, as this determines the dyes you can use. Also, not all cell sorters are built the same. Some instruments are very sophisticated and require a trained and dedicated individual to operate, while others were engineered for ease of use and are better suited for simple sorts (for example, sorting one or two fluorescent protein markers) (Brampton and Williams 2019).