Fluorescence-Activated Cell Sorting (FACS) is now a central component of many translational and clinical research applications that require pinpointing the phenotypes and genotypes of specific target cell populations. Whatever the downstream assay, there are some practical and experimental considerations that can help to optimize the overall success of a FACS sort (Arnold and Lannigan 2010). In this blog, we review some of the key steps and best practices formulated by the FlowMetric team of experts.

Garbage In- Garbage Out- Sample Preparation

As with all flow cytometry applications, the outcome is only as good as the cell preparation. For cell sorting, the preparation and maintenance of a single cell suspension is crucial to the outcome of the FACS run. Cell doublets or clumps will, at best, be eliminated from the sorting population and at worst result in a clogged instrument. We therefore filter all cell suspensions through a nylon mesh of 30-100µm frequently and at multiple steps and ensure that the cells are stored and sorted in an optimal buffer. For many cell types, this will mean Hank’s Balanced Salt Solution (Ca/Mg ⁺⁺ free) or a culture medium such as RPMI-1640 without phenol red, that is supplemented with 2% BSA or FBS (heat inactivated, and level adjusted to minimize autofluorescence of cells), and supplemented with 25mM HEPES pH 7.0, along with 1-5mM EDTA and 10U per mL of DNase II to help minimize cell clumping, all samples regularly agitated to prevent sedimentation. In most cases, cells do better when prepared and stored at 4˚C and kept cold during the sort, and the BD FACS Aria III is equipped with a recirculating water bath that supports the cooling of the collection chamber. All variables, from cell concentration, to buffer selection and storage temperature should be evaluated before the sort.

The preferred vessels for this type of cell work are made from glass, precoated with serum but if you need to use a single-use plastic tube, then round bottom polypropylene is the way to go. Avoid polystyrene, if possible, in order to minimize cell lost due to adherence of the cells to the tube walls.

The cell concentration of the pre-sort sample is somewhat driven by the stickiness of the cells and the speed of the sort. Lymphocytes are typically prepared at 20-30 x 10⁶ cells per mL, whereas cell lines are prepared at 5-10 x 10⁶ cells per mL.

Don’t Forget Your Controls!!

As with flow cytometry, FACS requires controls in order to ensure that a signal is real rather than an artifact of a lack of compensation or non-specific antibody binding. If you are targeting one of your markers will be dim or in low frequency, then antibody capture compensation beads may be a better option. This is critical for high complexity cell sorting that relies on multiparameter cell labeling for the identification of target cell populations. To this end, all antibodies should be titrated on the target cell population to optimize the signal to noise ratio and staining index are as high as possible. This will help to effectively discern the positive and negative populations with good separation. In addition, FMO controls are valuable both for gating as well as to determine the effect of spillover on a particular stain and should be employed when there is lower separation between unstained and stained populations (Perfetto, et. al. 2004). Finally, we highly recommend that a viability marker is used for all sorting sample. There is now a wide range of viability dyes available with different emission spectra so finding a reagent that is compatible with your fluorochromes should be possible. Examples of viability dyes that have been used at FlowMetric include: DAPI, 7-AAD, LiveDead, EMA and VivaFix. Note, that dyes such as 7-AAD and PI should be added just prior to sorting to prevent penetration into live cells over time; in contrast, amine dyes, that require a wash-step, are more forgiving in the timing between staining and sorting but are unable to stain cells that subsequently die during processing.

Optimizing the Instrumentation and Sorting Conditions

Set up for Aseptic Sorting

Prior to Aseptic Sorting it is important to clean sample path with 10% Bleach FACS for around 10 minutes followed by 1 minute of FACSRinse washing and 5 minutes of washing through sterile distilled water. The nozzle should be rinsed with 70% ethanol and airdried in a hood, and a fresh, autoclaved sheath tank should be used.

Wash your stained cell twice before FACS

Since population resolution is so crucial for FACS, the effective washing of unbound antibody/label is critical. At least two washes are recommended, and it is important to repeat cell counting after the last wash, for accuracy in cell concentration and optimal sorting performance.

Nozzle Selection and Speed of Sort

The upper limit for sorting rate is defined as the number of droplets formed per second, which is determined by the resonance frequency of the vibration of the liquid stream, driven by the nozzle size and velocity of the stream.

The nozzle selection is based on the size and sensitivity of the cell types to be sorted. Typically, the cell diameter is <1/3^rd of the nozzle size, although for very delicate cell types such as neural cells are best sorted at lower pressures and using larger nozzles.

The sorting conditions are a balance of three driving forces:

1. the final purity of the sorted population,
2. the yield of this cell population recovery from the cell preparation and
3. the time/speed taken to complete the sort.

These factors may require particular attention for the sorting of rare cell populations, when very starting sample sizes are processed over many hours to achieve target cell harvests. Positive or negative pre-sort enrichment can really help with sort efficiency and recovery. An example of this would be the sorting of naïve T-cells from spleen tissue; following harvest and dissemination of the spleen tissue. And RBC lysis step can be employed to remove RBCs, this can then be followed by CD4+ enrichment using the negative selection using MACS to deplete CTLs, B-cells, NK cells, Macrophages, Granulocytes, Erythroid Cells and DCs. The resulting cell population will be highly enriched in CD4+ cells, enabling the more efficient sorting out naïve T-cells.

Setting the Threshold

It is important for all sorting protocols, but particularly for purity sorts, that the FSC vs. SSC threshold be set appropriately to minimize the impact of debris on the sorting process. Setting the threshold to exclude the low-intensity signals from small debris particles will help in improving sorting yields and minimizing the occurrence of sorting aborts, where droplets are excluded from selection because debris coexists in a droplet with the target cell. If your downstream application requires the minimization of debris contamination of the sorted population(s), then the starting preparation should be as clean as possible, and the threshold should be adjusted for the instrument to ‘see’ debris particles; however, remember that this may impact several parameters of the sort.

Yield- the reality can be tough

There are inevitable losses of target cells from the cell sorting process. These losses can occur at all stages throughout the process from the staining/labelling centrifugation steps, to sorting abort events that discard droplets containing both a desired cell, and an undesired cell type. The bottom line is, the healthier your cell population going into a cell sort, the higher the viability of the sorted cell population. But it is important to consider that all of these stresses may have an impact on cell physiology and morphology. The most well described of these is the enhanced activation of p38 MAPK Activation in sorted T-cells (Andrä I et al. 2020). So downstream assay controls are always needed, and efforts to understand and address Sorter-Induced-Stress should be considered.

Fig. 1. Forces Imposed by Cell Sorting: The process of cell sorting can be very stressful for cells – with several parameters contributing to the impact on cells. There may therefore be considerable losses that may impact the overall yields.

Optimizing Collection

Other factors that can really help improve viable yields, include the dispensing of sorted cells into serum pre-coated recovery in tubes containing optimal media or serum. If sterility of the sorted population is needed, then the sample and collection media should be supplemented with 50µg/mL gentamycin.

Final Thoughts

FACS can support unprecedented target cell population enrichment; but investing time in panel optimization, sample pre-enrichment and post-sort viability support can make a profound effect on the yield, viabilities, and purity of the sorted population. Working with our experts at FlowMetric, can help ensure that your sorting needs are exceeded. Our Quality Management System and SOPs are in place to ensure all best practices for FACS are followed. Our experienced team have worked with challenging, and rare target cell populations, such as TILs and Mesenchymal Stem Cells, for downstream applications including RNA Seq, proteomic and metabolic profiling, or cell-based functional assays.

References

• Arnold, L. and Lannigan, J. Practical issues in high-speed cell sorting”, Current Protocols in Cytometry. 2010 Jan; Chapter 1: Unit 1.24.1-30.
• Perfetto, S., Chattopadhyay, P. & Roederer, M. Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4, 648–655 (2004). https://doi.org/10.1038/nri1416.
• Andrä I et al. (2020). An Evaluation of T‐Cell Functionality After Flow Cytometry Sorting Revealed p38 MAPK Activation. Cytometry A 97, 171–183.