Efficient cell pairing in droplets using dual-color sorting 2 3

The use of microfluidic droplets has become a powerful tool for the screening and manipulation of cells. However, currently this is restricted to assays involving a single cell type. Studies on the interaction of different cells (e.g. in immunology) as well as the screening of antibody-secreting cells in assays requiring an additional reporter cell, have not yet been successfully demonstrated. Based on Poisson statistics, the probability for the generation of droplets hosting exactly one cell of two different types is just 13.5%. To overcome this limitation, we have developed an approach in which different cell types are stained with different fluorescent dyes. Subsequent to encapsulation into droplets, the resulting emulsion is injected into a very compact sorting device allowing for analysis at high magnification and fixation of the cells close to the focal plane. By applying dual-color sorting, this furthermore enables the specific collection and analysis of droplets with exactly two different cells. Our approach shows an efficiency of up to 86.7% (more than 97% when also considering droplets hosting one or more cells of each type), and, hence, should pave the way for a variety of cell-based assays in droplets.

2B), the size of the restricted sorting channel was 40 µm × 40 µm (height × width) × 475µm (length).The main channels before the restriction channel were 75 um in height and width.
For the collection chip (Fig. 2F), the height of the low layer chamber was 40 µm and the size of the upper layer trap was 100µm in diameter × 100um in height.All microfluidic devices were fabricated using standard soft-lithography 3 .Molds were fabricated on silicon wafers using SU-8 resist (Microchem) and patterned by exposure to 375 nm light through 25400 dpi patterned masks (Suess).A mixture of 90% Polydimethylsiloxane (PDMS) elastomer (Sylgard 184 polymer base; Dow Corning) and 10% (w/w) curing agent (Dow Corning) was poured over the SU-8 molds, degassed and incubated at 65 degree overnight.Polymerized PDMS was peeled off from the mold activated by incubation for 1 min in an oxygen plasma oven (Diemer Femto) and bound to a 50 × 75 × 0.4 mm ITO glass (Delta Technologies).
Inlets and outlets were punched using 0.5 mm diameter biopsy punches (Harris Uni-Core) for electrodes and 0.75 mm diameter biopsy punches for the rest.The channels were first flushed by Aquapel (PPG Industries) and, subsequently, by HFE7500 oil (3M).

Cell cultivation and encapsulation
Her2 Hybidoma cells (ATCC® CRL-10463) were grown in complete DMEM medium (Gibco), Jurkat cells (ATCC® TIB-152) were grown in RPMI medium (Gibco), both supplemented with 10% FBS.Hybridoma cells were harvested, stained by Calcein-AM (Lifetechnologies) and Calcein Violet (E-bioscience), respectively, at room temperature for 45 min, washed by PBS twice to remove free dye in the media , and re-suspended in free style media (Gibco) supplemented with 1 mg/ml xanthan gum (Sigma) to prevent cell sedimentation during encapsulation.Subsequently, green and violet cells were mixed equally at a final concentration of 1.5 × 10 6 cells/ml and injected at a flow rate of 1000 µl/h into the droplet generation chip.Droplets were generated by flow focusing this continuous phase using Novec HFE7500 oil, containing 5% PEG surfactant 3 (custom synthesized at Sigma Aldrich), at a flow rate of 4000 ul/h.Emulsions were collected in a collection tube (cryotobube, Nunc) which was treated with Aquapel (PPG industries) and, subsequently, rinsed by HFE7500 oil.

Sorting and Imaging
Emulsions were re-injected using an electro-osmotic pump (Nano Fusion Technologies) at a flow rate of about 60 µl/h.Oil with 0.5% and 0.25% of PEG surfactant were loaded in syringes individually and injected by Harvard Apparatus PHD 2000 syringe pumps at a flow rate of 400 µl/h (Fig. 2B, (a)&(c)) and 600µl/h (Fig. 2B, (d)) respectively .A refilling pump was connected with outlet E (Fig. 2B, (e)) to withdraw all of the droplets that did not trigger sorting to the waste syringe at a flow rate of 760 µl/h.Droplet sorting videos were acquired at ~500 frames per second.A customized LabVIEW sorting program was used to control the droplets sorting.The positive droplets were collected in the collection chip (Fig. 2F-H) and the trapping events were monitored on a cell imaging device (CytoMate Inc.).The collection was finished when all of the traps were occupied.Subsequently, the collection chip was rinsed with oil containing 0.25% PEG surfactant to remove un-trapped droplets.
Sorting enrichment was determined by automated scanning of the entire collection chip at 10-fold magnification using an inverted fluorescence microscope (Nikon eclipse Ti), equipped with a motorized stage and a Hamamatsu Digital camera.
82 Table S1 S1.Flowchart summarizing the logic of the LabVIEW control software programmed at EMBL, Heidelberg.This algorithm runs in parallel for both of the PMT channels (one for each colour) and detects peaks in the signal values.This allows cells within droplets to be detected and for a sorting decision to be made for each passing droplet based on the intensity of the signal, the number of peaks detected, the width of the overall peak and the spacing between droplets that contain at least one cell.This software and a user manual can be freely downloaded for academic use at www.merten.embl.de/index.html.Imaging is performed using an inverted microscope equipped with a high speed camera.

Fig. S3
. Example of the signal peaks in one droplet.The zoom in (inset) reveals a jigsaw shape of the signal at low intensity, thus making the use of inflection points for the detection of peaks impossible.

Fig. S4. Signal variation of Calcein-AM and Calcein-violet stained cells inside droplets. (A)
Droplet showing one green peak and two overlapping violet peaks, corresponding to a clump of 2 violet cells, with a valley between the two peaks above a value of 0.5 fluorescence units.This value is higher than the green peak of another droplet (B) hosting exactly one green and one violet cell.Therefore using static thresholds (solid black lines) is not sufficient to accurately detect the number of encapsulated cells.However, when specifically detecting drops in the fluorescence signal exceeding the maximum noise (red dots), the number of peaks can be correctly determined, independently of the peak intensities.

Fig. S2 .
Fig. S2.Schematic of the optical setup.The fluorescence-based sorting setup uses diode lasers with excitation wavelengths of 405 nm (Calcein Violet), 488 nm (Calcein-AM) and 561 nm (optional third laser for assay readouts).Emission signals are detected using PMTs with a 450 nm band-pass filter (blue), a 521 nm band-pass filter (green), and a 610 nm longpass filter (red).Sorting signals are processed using LabVIEW software running on a FPGA card triggering a high voltage amplifier.Imaging is performed using an inverted microscope equipped with a high speed camera.

Figure S5 .
Figure S5.Leakage of Calcein Violet from cells encapsulated into droplets.(A) Zoom in of the fluorescence signals over 8 hours incubation at room temperature.The strongly decreased scale of the Y-axis (from 0 to 0.05 A.U.) allows illustrating the increase in the droplet signal (wide peaks), but requires cropping of the cell signals (narrow subpeaks with intensities as shown in (B)).(B) Time course of fluorescence signals of droplets hosting Calcein Violet-stained hybridoma cells.After incubation for the indicated time periods off-chip, the droplets were reinjected into the sorting device and the fluorescence signals were determined in the detection channel using a PMT.. (C) Fitted LOESS smoothing line of droplet fluorescence intensities (turquoise line), individual data points (turquoise circles) and confidence (grey shades) of the droplet signals.(D) Fitted LOESS smoothing line of cell fluorescence intensities (red line), individual data points (red circles) and confidence bands (grey shades) of the cell signals.(E) Intensities of cell and droplet signals plotted at the same scale.

Fig. S7 .
Fig. S7.Efficiency of the sorting process for droplets hosting differently stained Her2 Hybridoma cells.Blue fluorescence of droplets captured in the collection chip before (A) and after (C) sorting.Green fluorescence of droplets captured in the collection chip before (B) and after (D) sorting.

Fig. S8 .
Fig. S8.Fluorescence analysis of the droplets detected by PMT.(A) Two dimensional dot plot of fluorescence signals of droplets.The red arrow indicates one example of the dual color droplet with two cells.(B) Dot plot showing violet and green signals of the droplets.(C) Droplet occupancy before sorting .

Fig. S9 .
Fig. S9.Efficiency of the sorting process for droplets hosting differently stained Jurkat cells.Blue fluorescence of droplets captured in the collection chip

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Sorting results.ND = not detectable