APPLICATION NOTE

Mixed-mode Sorting with CytoFLEX SRT Cell Sorter
Objective• Define common sort terms.
• Learn about the different sort modes available on the CytoFLEX SRT.
• Understand the utility of mixed-mode sorting.
Introduction
Flow sorters can physically separate a desired population of cells from a cell suspension. They combine
a sensitive flow cytometric analysis with a sorting apparatus. The analysis components establish cell
identity by presence or absence of surface or intracellular proteins. Cells can also be more finely divided
based on activation status and expression of protein by analyzing or sorting based on a spectrum ofexpression. Once a target population is selected, software and the sorting apparatus divert a droplet
containing the particle of interest to the desired output vessel. Sorting can provide a highly enriched
population of cells for downstream use in a wide variety of assays, such as genomics, adoptive celltransfer, culture, proteomics, and imaging.
The CytoFLEX SRT is an electrostatic droplet sorter. The sorting apparatus that is used is similar to thesystems that make inkjet printers work. In the CytoFLEX SRT, after cells are interrogated in the flow
cell, they exit the flow cell in a stream. That stream receives vibration energy from a vibrating crystal
associated with the flow cell. This vibration causes the stream to break up into droplets as seen in
figure 1. The frequency of this vibration is how fast droplets are made and this value can vary slightly
from day to day. Once an event of interest is detected, the system waits until that event is in the lastattached droplet on the stream, the stream is then charged in time with the vibration frequency so thatthe droplet breaks off with a static charge and the rest of the stream returns to ground potential. Whenthe charged droplet passes through a set of charged plates, it is pulled toward the plate with opposite
charge and lands in the collection vessel.

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Figure 1. An illustration of Droplet Formation. Note the frequency in Hertz, which is also the rate of droplet formation. The last attacheddroplet is directly above the red line and is the droplet targeted to receive an electrostatic charge.

This application note focuses on the different sort modes available on the CytoFLEX SRT cell sorter.
Sorts may have different desired purity or yield outcomes based on the downstream assay. This canbe addressed through the selection of an appropriate sort mode. This application note defines anddescribes the different sort modes on the CytoFLEX SRT cell sorter. Some common sort terms will also
be defined.

Common Sort Terms
Sort Logic
Combination of Boolean (e.g. AND, OR, or NOT) or hierarchical logic of regions and gates to determine
if an event is positive (i.e desired for sorting). Events that fall outside the sort logic are considerednegative events. Depending upon the sort mode, negative events can be considered contaminants
and can cause a positive event to be aborted. Regions are defined in terms of either one parameter(histogram) or two parameters (dot plot). Gates are Boolean combinations of regions. Hierarchical gatesare created from nested populations – a population within a population within a population. CytoFLEX
SRT creates both Boolean and Hierarchical gates. The user defines which gate to apply to the sort logic.

Sort Mode
A command given to the software to establish when to charge and deflect a drop. The sort mode plays
a crucial role to determine the ultimate yield and purity of the sorted cells based on the proximity ofcontaminants (non-targets) to the particle of interest as selected using the sort logic.

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Sort Envelope
The sort drop envelope defines how many drops will be sorted based on the position of the positive
event in the drop. To make decisions on envelopes easier, CytExpert SRT has several pre-defined mode/envelope combinations for common practices. (For more on sort envelopes review the “CytoFLEX SRT
Sort & Stream Precedence” module in the Beckman Coulter Learning Center.

AbortAn event that met the requirements in sort logic but had a contaminant in the sort Guard Band (i.e. tooclose to allow for sorting based on the sort mode). Often these abort events go into the waste stream
and cannot be recovered. However, with CytoFLEX SRT these events can be sent to an alternative
stream to allow for culturing, re-sorting or further analysis.

Efficiency
A measure of how many events were aborted. Expressed as a percentage it is calculated by (Dropssorted)/(Drops sorted + Drops aborted) x 100%. The higher the percentage, the fewer aborted eventsin the sort. When running on clean single-cell suspensions, efficiencies of over 90% can be achieved.
Clumps, higher event rates, and debris in the sample can all decrease efficiency.

RecoveryBased on the estimated number of cells that were reported as sorted, recovery is the percent that arecounted in the tube. It can be estimated by several methods such as visual counting or running a sample
with volumetric counting beads.

Yield
Based on the estimated number of cells in the starting sample, yield is the percent that were sorted andreturned for downstream use. Formulated as Yield= ((# of events in the collection tube x purity) / (total
number of particles in the starting tube x percent target particles)) x 100%.

Sort Modes Defined
Selected populations may be sorted using three different modes depending on the output desired.

Enrich Mode
Enrich Mode is used when recovery is the most important aspect of the sort. With Enrich,
all positive events (i.e. events that satisfy the sort logic) are sorted.

• Used when recovery is more important than purity.
• All sort logic events are sorted, regardless of any negative events (i.e. an event that falls outside thesort logic) in or near the drop being sorted.

• No aborts present using this mode due to negative events or hardware coincident events, therefore
efficiency will always be 100%. (Drops sorted)/(Drops sorted + Drops aborted) x 100%

• Provides the greatest efficiency and recovery but least purity. Purity decreases as the event rateincreases and approaches the droplet generation rate.

Purity Mode
Purity Mode is used when the purity of the sort is the most important aspect. Negative events withclose proximity to positive events make sorting a pure droplet unlikely and the droplet containing thepositive event is sent to the stream indicated for aborts (e.g. waste or another sort stream).

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Used when purity is more important than recovery.

• The drop being sorted will contain only events that fit the sort logic.
• If the drop contains negative events, it will be aborted.
• If the nearest edge (15%) of an adjacent drop contains any contaminating events, the primary drop
will be aborted. In CytoFLEX SRT this edge percentage is called the Sort Guard Band and can be
adjusted to be more stringent if desired.

• As the event rate increases, the abort rate will also increase because of the likelihood that theprimary or near edge of an adjacent droplet will have a negative event.

Single Mode
The Single Mode is used when having only one positive event per drop is the most important aspect ofthe sort. It is most commonly used in plate sorting for single cell applications such as cloning and -omics
applications.

• Having one positive event per droplet is the most important aspect of single mode.
• Recovery is lower, but purity remains high.
• Only a single positive event and no negative events will be in the drop sorted.
• If the nearest edge (15%) of an adjacent drop contains any positive or negative events, the primarydrop will be aborted.

• As the event rate increases, the abort rate will also increase because of the likelihood that theprimary or near edge of an adjacent droplet will have either a positive or negative event.

• Used most often for single cell deposition into plates/slides for cloning purposes.
Mixed-mode Sorting Cases
Using Purify and Enrich Mode Sorting
Mixed-mode sorting can allow a high level of flexibility to your sort experiments. One example is a casewhere the sort requires enrichment of a rare population that cannot be lost to aborts due to expense,
rarity of sample, or other constraints. We will stain a process control product with a multicolor paneland demonstrate the effect of different sort modes on the sorted cell population outcomes. Figure 2
shows staining of the unsorted sample.

Figure 2. Sample Analysis Prior to Enrich and Purify Sorts. Reconstituted lyophilized human lymphocytes, CYTO-COMP Cell Kit (PartNumber 6607023) were stained with DURAClone IM T Cell Subsets (Part Number B53328) per the instructions for use and data acquiredon the CytoFLEX SRT Cell Sorter. Regions were drawn using CytExpert SRT software. Population statistics are displayed for all regions
for comparison to post sort populations.

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The CytoFLEX SRT cell sorter has been set up to enrich for CD8+ T cells, figure 3. This sample could
now be run at high speed compared to the rate of droplets generated and minimize cell loss of thispopulation. Note the high efficiency of this sort indicating no loss due to aborts. Upon re-analyzing thesorted sample, it can be shown that purity is still higher than in the initial sort sample, but lower thanexpected if using a more stringent sort mode such as Purify, figure 4.

Figure 3. Tube Sort Statistics for CD8+ Enrich Mode. Tube Sort window showing settings for four sort streams as gated on the sample
shown in figure 2. R1 position is set to Enrich mode for CD8+ cells. Red boxes indicate zero aborts and 100% sorting
efficiency, respectively.

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Figure 4. Posts-sort Analysis of CD8 Enriched Cells. The R1 stream was analyzed post sort. CD8+ cells have been enriched representing
95.33% of total cells versus 24.89% of the starting sample (figure 2). The sorted cells still contain CD4+ cells.

In the same experiment the researcher wanted the Effector population to have high purity. The sortmode selected in this case is Purity, figure 5. Notice the lower efficiency in this stream.

Figure 5. Tube Sort Statistics for Effector Purity Mode. Tube Sort window showing settings for four sort streams as gated on the sample
shown in figure 2. L1 position is set to Purity mode for CD4+ Effector cells. The red box indicates the lower efficiency statistic for this sort
as compared to the Enrich mode.

Every Event Matters | 6Upon re-analysis of the sorted product (figure 6), the population sorted with the Purify mode is at avery high purity compared to the population sorted with the Enrich mode. The tradeoff however is thatthe sort is overall less efficient as some cells are lost to aborts. This tradeoff between purity and abort
rate becomes more evident when sorting at higher speeds as the events per second approach the rateof droplet generation.

Figure 6. Post-sort Analysis of Effector Cells Sorted for Purity. The L1 stream was analyzed post sort. CD4+ Effector cells now
representing 98.21% of total cells versus 2.01% of the starting sample (figure 2). The sorted cells do not contain CD8+ cells.

Sorting Populations for High Purity at High Speed
We’ve previously shown that sorting at higher speeds compared to your rate of droplet formation
results in lower efficiency (i.e. higher aborts). In this section we will show that by making use of mixedmode sorting, we can sort populations to high purity in one stream and send aborts from that and othersort streams to another stream. In this way, aborted events can be used for other experiments rather
than sent to waste.

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Figure 7. Sample Analysis Prior to T Cell Memory Sorts. Reconstituted lyophilized human lymphocytes, CYTO-COMP Cell Kit (PartNumber 6607023) were stained with DURAClone IM T Cell Subsets (Part Number B53328) per the instructions for use and data acquiredon the CytoFLEX SRT Cell Sorter. Regions were drawn using CytExpert SRT software. Population statistics are displayed for all regions
for comparison to post sort populations.

Shown below is a sort for CD4 T cell memory subsets from the sample analysis shown in figure 7. ThePurity sort mode was used for all three target populations, and the aborts were sent to sort stream R2,figure 8.

Figure 8. Tube Sort Statistics for Purity Mode Sorts and Abort Catches. Tube Sort window showing settings for four sort streams asgated on the sample shown in figure 7. L2, L1, and R1 positions are set to Purity mode and R2 is set to catch aborts from the three otherstreams.

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As long as one of the four streams is unused, aborted droplets from any or all of the other streams canbe sent to this alternative, allowing for lossless sorting of target populations. Figure 9 displays the post
sort analysis of the abort stream. It has representation of all the selected events, plus the undesired
contaminates that contributed to the aborts.

Figure 9. Tube Sort Statistics for Purity Mode Sorts and Abort Catches. Tube Sort window showing settings for four sort streams as gatedon the sample shown in figure 7. L2, L1, and R1 positions are set to Purity mode and R2 is set to catch aborts from the three other streams.

Tips for success• When sorting you are balancing speed, yield, and purity. A researcher can typically emphasize twoof these at a time at the expense of the other one.

• Abort streams can be helpful for troubleshooting if there is a contaminating population that iscausing most of your aborts. Re-analysis of abort streams can also show flaws in your gating
strategy.

• Abort streams also allow for retention of the population of interest, even if it is contaminated.
This sample can be re-sorted to a higher purity of desired.

• Remember that you can combine abort streams into one stream or separate them into individual
abort streams.

• Enrich sorts have no aborts.
Summary
With multiple options for sort mode, the CytoFLEX SRT allows researchers to have a high level ofcontrol over the level of enrichment of targets required on a case-by-case basis. This mixed-mode utility
can allow for lossless sorting of valuable populations. Populations can be sorted to high purity, while
preserving aborted events for re-sorting or further analysis.

* For research use only. Not for use in diagnostic procedures.
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