Getting the most from your cell sorter

Fluorescence-activated cell sorting (FACS) is an increasingly common step in many life science research applications, from sequencing to protein design to immunotherapy. No matter which downstream application is used, it is possible to take steps to optimize the purity, efficiency, and reliability of each sort performed.

At a webinar, Flow Cytometry Laboratory Directors Peter Lopez, PhD, of NYU Langone and Rui Gardner, PhD, of Memorial Sloan Kettering Cancer Center presented a set of best practices for cell sorting. Their recommendations about best practices spanned the process from experimental planning and choosing a sort mode, through sample preparation and setup, to monitoring the sort, and post-sort analysis.

Here, a few of their considerations are summarized. The webinar is available for replay for additional comprehensive advice. Both laboratories use cell sorters from Sony Biotechnology. Automation and intuitive software features make them ideal for researchers new to cell sorting. Yet the sorters are advanced, reliable, and flexible enough to satisfy experts.

 

Improving single-cell suspension

The success of cell sorting using flow cytometry depends on the cells being separated from each other as they pass through the flow cell. Dr. Gardner presented five sample preparation tips for improving single-cell suspension and avoiding doublets (Figure 1) and aggregation. For example, dead cells in the suspension will release DNA, which promotes clumping. It can be reduced by adding DNAse. A microscope can be used to inspect for clumps and dead cells, which can then be removed with the appropriate filtration. For lengthy sorts, prevent sedimentation and clumping by using a sample agitator or mixing the samples by hand.


Figure 1. Five insider tips that improve your single cell suspension

Setting up and monitoring the sort

Dr. Gardner also emphasized the importance of calibrating the streams into the collection tubes or plates and monitoring the sorter during operation. “Don’t go for lunch while the sorter is doing its job,” he cautioned. He also advised monitoring the number of events per second and the number of non-sort events (aborts).

Advanced automation can handle several of these steps for you. Sorters from Sony, for example, offer automated optical axis adjustment, automated side stream calibration, automated drop delay calibration, and automated sort monitoring and droplet calibration. These features allow detection of clogs and empty tubes and enable walkaway operation if you so choose.

 

Post-sort re-analysis


Figure 2. Post-sort re-analysis.

Once the sort is complete, Dr. Lopez advised re-analyzing the sorted cells to verify the purity of your cells of interest—measured by how much unwanted material ends up in the sorted fraction. Also, verify that cells are behaving as intended. If you use the cell sorter to re-analyze the sorted population, make sure to clean thoroughly (and on sorters from Sony, replace the fluidics chip) beforehand to minimize carryover from the initial sort, because any carryover will resemble the original unsorted population.

Figure 2 shows re-analysis of a sorted population that has been gated to exclude debris (A), doublets (B), and dead cells (C). From the PEhigh cells (D), two populations were originally sorted (R5 and R8). Panel E shows re-analysis of the R5 fraction. With very few unwanted cells (empty R8 region), this represents a high-purity sort. However, notice that the population has drifted slightly toward the right of the expected R5 region. It is legitimate to redraw the region to encompass what is clearly a single population.

 

Minimizing SICS

At CYTO 2018, Dr. Lopez introduced the term sorter-induced cell stress (SICS) to represent the cellular damage that might be caused by cell sorting. Although SICS does not always occur, effects can range from functional changes to cell death. Table 1 outlines Dr. Lopez’s methods to minimize SICS, starting with using a larger nozzle (and on sorters from Sony, the appropriate size sorting chip). If all these methods are unsuccessful, you might need to use a different sorting technology.

 

The Sony solution

Size of Orifice Sorter Temperature
Use a larger orifice (implies lower sheath pressure) Cells may do better when sorted using instrumentation set at 4°C or room temperature
Sorting Buffer Duration of the Sort

Collecting cells in media with high serum content or even 100% serum

Consider the dilution effect coming from the sheath fluid on the total collection volume

For genomics applications, collect sample directly in genomics lysis buffer

Break up sample into smaller aliquots for sorting

Remove sorted fractions off the sorter at regular intervals

Get sorted cells into their most favorable conditions (e.g., buffer)

Table 1. Ways to minimize sorter-induced cell stress.

Sony Biotechnology offers a line of advanced cell sorters that can help you sort cells successfully every time for a wide range of downstream applications. Their microfluidics-based sorting chips, automated calibration and cleaning cycles, and other tools and options make it easy to apply these best practices. All sorters feature process automation, intuitive software, and standardized calibration to ensure consistent results from day to day and instrument to instrument.

Sony’s newest sorter, the MA900 Multi-Application Cell Sorter, offers Sony’s highest level of automation yet, including sort setup, QC, and aseptic cleaning. It features up to 12 colors, 14 parameters, and 4-way sorting into tubes or plates. Advanced controls provide flexibility to accommodate a wide range of applications.

 
Learn More About the MA900 Multiapplication Cell Sorter from Sony →

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