Reliable Single-Cell Sortingwith Bio-Rad’s S3e™ Cell Sorter
Joyce Lee
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547

Cell Sorting

Bulletin 6853

Abstract

Accurate isolation of individual cells is essential for single-cell analysis techniques. Here, we
demonstrate a simple workflow to verify the reliability of the S3e Cell Sorter in sorting singlecells. Using this method, we confirmed sorting accuracies of >94% and 100% for single cells
and single beads, respectively.
Introduction
Cellular heterogeneity, originating from stochastic gene
expression, leads to difficulties in interpreting cell-to-cellvariations in transcription and translation (Raj and vanOudenaarden 2008). Therefore, it is important to examine
genomic, epigenomic, and transcriptional variation at thesingle-cell level. Flow cytometry combined with single-cellsorting allows researchers to identify and separate target cells
for cell culture and other downstream analyses such as gene
expression, transcriptome sequencing, and cell imaging. In
this study, we demonstrate a method to verify the accuracy of
single-cell sorting using the ZOE™ Fluorescent Cell Imager as an
essential QC step and thus confirm that the S3e Cell Sorter iscapable of reliably sorting single cells into 8-well culture strips.
Materials and Methods
Preparation of Jurkat Cells and Beads for Sorting

Jurkat cells (ATCC, catalog #TIB-152) were grown in RPMI
media supplemented with 10% FBS and passaged every
2–3 days to ensure viability. Before sorting, the cells werewashed twice with sort buffer (3% FBS in PBS), counted with
the TC20™ Automated Cell Counter (Bio-Rad Laboratories,#1450102), resuspended to a final concentration of2 x 106 cells/ml, and split evenly into two 5 ml tubes. The
unstained control cells in the first tube were filtered througha 40 µm strainer and used to set up gating and voltage
parameters on the S3e Cell Sorter. The Jurkat cells in thesecond tube were stained with CytoTrackTM Green 511/525
Dye (Bio-Rad, #1351203). The appropriate volume of 500x
CytoTrack stock solution was added to the cell sample to
make a 1x final concentration and the sample was incubatedin the dark at room temperature (RT) for 15 min. The cells werethen centrifuged at 1,000 x g for 5 min at RT, the supernatant
was discarded, and the cells were washed with 3 ml sortbuffer. The cells were then resuspended in 1 ml of fresh sortbuffer and filtered through a 40 µm cell strainer prior to loadingon the S3e Sorter. For the bead sorting experiment, 30 µl of
15 µm polystyrene bead (Thermo Fisher Scientific, #F13838)
were added to 500 µl PBS in a 5 ml tube.
S3e Cell Sorter and Cell Sorting Setup

We used an S3e Cell Sorter equipped with a blue (488 nm)
argon laser and a red (640 nm) solid-state laser(Bio-Rad, #1451008). The drop delay was calculated by directly
counting the ProLine™ Universal Calibration Bead(Bio-Rad, #1451086) events in the waste stream during thequality control process. In order to ensure optimal sorting
conditions into the 8-well flat bottom culture strips (Corning,#9102), deflection values were evaluated and adjusted asnecessary. One 8-well strip was placed onto a left-side 8-wellflat bottom S3e Collection Adaptor (Bio-Rad, #1441011) andfive ProLine Universal Calibration Beads were sorted into
each of the 8 wells. The wells were then visually examined tocheck the position of the sorted beads at the bottom of thewells. Deflection charge settings were adjusted until the sorted
droplets landed at the center of each well. The location of thedroplet in each well was marked and used as a template for
future sorts.
Single-Cell Sorting on the S3e Cell Sorter and Verification
with the ZOE Fluorescent Cell Imager

Prior to single-cell sorting, 1 µl of mineral oil (Sigma, #M3516)
was added to the center of each well of the 8-well strip. This
small amount of mineral oil was used to maintain the sortedcells or beads to a confined area for easy visualization andconfirmation of single-cell sorting. (Note: this method is notrecommended for other sorting applications or general S3e
Sorter use). The cells or beads were then sorted into the 8-well
strip. To prevent sample evaporation, the strip containing thesorted sample was placed on a rack and immediately
covered with a lid. The number of cells or beads in eachReliable Single-Cell Sorting with Bio-Rad’s S3e ™ Cell Sorterwell was verified using the ZOE Fluorescent Cell Imager.
Cell images were captured in both brightfield and green
fluorescence channels, while only brightfield images werecaptured for beads.
Results
Optimization of deflection values is crucial for sorting
accuracy. Appropriate deflection settings ensure that thesorted cells are directed to the correct position in the 8-well
strip in order to optimize recovery. Ideally, sorted cells aredirected to land as close as possible to the center of the well.
Therefore, test sorts were performed into each well of the8-well strip. Charge Left and Charge Right deflection values
were adjusted by 1% for each side (left and right) and thetest sort was repeated until the best deflection values wereidentified (Figure 1). The ideal sort deflection resulted in all
sorted drops landing at the center of the wells in the 8-wellstrips. For all experiments described, the S3e Cell Sorter
consistently sorted cells or beads into each well of the 8-well
strip. The S3e Sorter performed sorts to either the left or theright collection points with equal accuracy.

The accuracy of sorting single cells and single beads wasverified using Jurkat cells stained with CytoTrack Green
511/525 Dye and 15 µm polystyrene beads, respectively.
CytoTrack Dyes label live cells efficiently and allow easy
visualization and verification of single sorts using the ZOE
Fluorescent Cell Imager. Images of single sorted cells andbeads are shown in Figure 2. Both cells and beads were
sorted in groups of 13 strips for a total of 104 wells each. Of
these, 98 contained only a single cell. The remaining wells
were found to have no fluorescence signal and contained only
cellular debris. For the 15 µm bead sorting application, all 104
wells contained only a single bead. Therefore, we calculated
the accuracies for single-cell and single-bead sorting
applications as >94% and 100%, respectively.
A
1X (BF)

1X (G)

1X (M)

4X (BF)

4X (G)

4X (M)B
1X (BF)

4X (BF)

Fig. 2. Verification of single-cell and -bead sorting using the ZOE
Fluorescent Cell Imager. Visualization of a single Jurkat cell (A) and 15 µmbead (B). Magnification power: 1X and 4X. BF, brightfield; G, green fluorescencechannel; M, merged brightfield and green channel.

Fig. 1. Deflection value optimization for collection of single cells in 8-wellflat bottom culture strips. The Edit Streams Setting (Streams Control window)
is found under the Administrator Toolbar. The Charge Left and Charge Rightdeflection values were adjusted in 1% increments until the test sorts using
ProLine Universal Calibration Beads confirmed that the sorted events weredirected to the correct position within the 8-well strips.

© 2016 Bio-Rad Laboratories, Inc.

Conclusions
We have demonstrated a simple workflow to verify that theS3e Cell Sorter consistently and reliably sorts single cells
into each well of 8-well flat bottom culture strips (Figure 3).
The accuracy of sorting single cells and beads was verified
using the ZOE Fluorescent Cell Imager. No wells contained
more than one cell or bead. While the data presented here
illustrate the inherent precision of the S3e Cell Sorter, it isrecommended that each lab run this workflow to optimize thesystem for their cell type or application.

Bulletin 6853

Reliable Single-Cell Sorting with Bio-Rad’s S3e ™ Cell Sorter1 Count

2 Stain• Harvest cells and wash twicewith sort buffer• Stain cells with CytoTrack Green
511/525 Dye and incubate in thedark at RT for 15 min• Count cells with TC20
Automated Cell Counter andresuspend in sort buffer to a
final concentration of
2 x 10 6 cells/ml

3 Sort• Pellet the cells, wash the cellpellet with sort buffer, and thenresuspend in 1 ml sort buffer

4 Image/Verify• Adjust the deflection charge setting on the S3e Cell Sorter
using ProLine Universal Calibration Beads• Add 1 µl of mineral oil to the center of each well of the8-well flat bottom culture strip• Capture cell/bead images with the ZOE Fluorescent Cell Imager		– Cell: brightfield and green flourescence channels		– Bead: only brightfield channel• Sort cells and beads into 13 strips (104 wells) each• Calculate the accuracies for single-cell and single-bead sorting

Fig. 3. Single-cell sorting verification workflow. Depiction of a simple workflow to verify the accuracy of single-cell sorting on the S3e Cell Sorter.
The ZOE Fluorescent Cell Imager serves as the final QC/verification step.

ReferencesRaj A and van Oudenaarden A (2008). Nature, nurture, or chance: stochastic gene expression and its consequences.
Cell 135, 216–226.

© 2016 Bio-Rad Laboratories, Inc.

Bulletin 6853

Reliable Single-Cell Sorting with Bio-Rad’s S3e ™ Cell Sorter

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