DOI: 10.5061/dryad.wstqjq2zr
title: Data from: Deterministic cell pairing with simultaneous microfluidic merging and sorting of droplets

description: Cell–cell interactions drive immune activation, tissue repair, and stem
 cell fate, yet there are few methods that can create large numbers of
 pre-defined cell pairs to study cell crosstalk. Droplet microfluidics
 allows high-throughput compartmentalization of multiple cells, but random
 loading results in &lt; 1 % of droplets containing the desired
 combinations. Here, we present Pair Isolation by Coalescence and Sorting
 (PICS), a microfluidic platform that can generate specific cell pairs
 through droplet merging and sorting (‘merge-sorting’). PICS detects target
 combinations using fluorescence and triggers simultaneous
 electrocoalescence and dielectrophoretic sorting. Using fluorescent
 dye–loaded droplets, we achieved 98.6 % purity of merged and sorted
 droplets. In experiments using cells stained with three distinct dyes,
 &gt; 90 % of desired cell pairs were recovered – compared to fewer
 than 1 % when using random Poisson loading. To demonstrate the utility of
 PICS for extended co-culture studies, we merged cells in an alginate
 solution with calcium chloride droplets, producing monodisperse alginate
 hydrogels in which 93.3 % of the beads contained target cell pairs that
 maintained viability over 18 hours. Compared to selective merger, this
 approach physically isolates desired droplets, eliminating unmerged
 contaminants and enabling cleaner downstream workflows. PICS allows
 off-chip pre-incubation of droplets before pairing, the merger of reagents
 for multi-step assays, and the rapid isolation of desired droplet pairs –
 capabilities not jointly accessible with existing approaches. In summary,
 PICS is a flexible platform to enrich specific combinations of droplets,
 cells, or particles for high-throughput studies of cell crosstalk.
description: Droplet microfluidic images and videos were collected using an
 AE31 inverted microscope and a Phantom Miro M110 high-speed camera.
 Droplet/gel collection images were collected on a Leica DMi8 inverted
 microscope using the brightfield and Leica GFP (11525314), Y3 (11525311),
 and Cy5-naEx (11536078) filter cubes to detect FITC/Calcein Green, Cell
 Trace Yellow, and Cell Trace Far-red, respectively. Length scale
 information was determined from the automatically generated scale bar from
 the Leica DMi8. Individual microscope image channels were then imported to
 ImageJ, where fluorescent intensity, droplet/gel size, cell pairing, and
 viability data were analyzed using the software plugins. The analyzed data
 were then input to GraphPad Prism to generate data visualizations. Device
 geometry was drafted in Autodesk AutoCAD. Additional experimental methods
 description can be found in the associated manuscript.
description: # Data from: Deterministic cell pairing with simultaneous microfluidic
 merging and sorting of droplets Dataset DOI:
 [10.5061/dryad.wstqjq2zr](10.5061/dryad.wstqjq2zr) ## Description of the
 data and file structure This dataset was used to generate the results and
 figures included in "Deterministic cell pairing with simultaneous
 microfluidic merging and sorting of droplets" (Joslin et al., 2025).
 The dataset consists of microscopy images, included as TIFF (.tif) or JPEG
 (.jpg) files, and AutoCAD drawing files (.dwg). More details on data
 collection can be found in the Methods section of this dataset and the
 manuscript.  ### Files, folders, and variables #### File:
 data_from_Joslin_et_al_2025.zip **Description:** Zip file for complete
 data for "Deterministic Cell Pairing with Simultaneous Microfluidic
 Merging and Sorting of Droplets" (Joslin et al., 2025). Contains the
 folder *data_from_Joslin_et_al_2025*, which contains subfolders organized
 in the following structure: * data_from_Joslin_et_al_2025 *
 Device_Geometry * Fig1 * Fig2 * Fig3+FigS6 * Merge-sort_All *
 Merge-sort_Cell1&2 * Merge-sort_Cell1&3 * Merge-sort_Off *
 Fig4+FigS7 * K562_Starting_Calcein_Green * Merge_All * Merge-sort *
 Selective_Merger * FigS1 * FigS2 * FigS3 * Collection * Snapshots * FigS4
 * FigS5 * drops_in_sort_area * merge-sort_all * merge-sort_off *
 merge-sort_peak_duration_gate #### Folder: Device_Geometry
 **Description:** Contains AutoCAD drawing files (.dwg), Drawing Exchange
 Format (.dxf) files, and Scalable Vector Graphics (.svg) files for the 40
 um drop maker used to generate input emulsions,
 *40um_drop_maker.dwg*, *40um_drop_maker.dxf,* and *40um_drop_maker.svg,*
 and the PICS device used to merge-sort droplets,
 *PICS_device.dwg*, *PICS_device.dxf,* and *PICS_device.svg*.  #### Folder:
 Fig1 **Description:** Data supporting figure 1 of the manuscript. Contains
 6 fast camera snapshot image files (*.tif*) when the merge-sort device is
 OFF (*merge-sort_OFF_frame_**X1**.tif*) and ON
 (*merge-sort_ON_frame_**X1**.tif*), where ***X1*** is *1-6*, denoting the
 chronologic position of the snapshot, with *1* being the beginning and *6*
 being the end. An example file name is *merge-sort_ON_frame_6.tif*, which
 is the sixth frame in a short time course of the droplets in the PICS
 device when merge-sorting is turned on.  #### Folder: Fig2
 **Description:** Data supporting figure 2 of the manuscript. Contains
 fluorescent microscopy images (*.tif*) of the input emulsion
 (*input_emulsion_**X2**.tif*), collected emulsion when merge-sorting was
 OFF (*merge-sort_OFF_**X2**.tif*), and collection emulsion when
 merge-sorting was ON (*merge-sort_ON_**X2**.tif*). ***X2*** is *ch00* for
 the bright field image channel, and *ch01* for the FITC image channel.
 These images were used to generate figure 2. An example file name is
 *input_emulsion_ch01.tif*, which an image of the FITC channel for the
 input emulsion used in figure 2.  #### Folder: Fig3+FigS6 **Description:**
 Data supporting figures 3 and S6 of the manuscript. Contains
 folders *Merge-sort_All, Merge-sort_Cell1&2, Merge-sort_Cell1&3*,
 and *Merge-sort_Off* which respectively contain fluorescent microscopy
 images (*.jpg*) of collected emulsions for merge-sort conditions where all
 drops where merge-sorted, only cell1&2 containing drops were
 merge-sorted, only cell1&3 containing drops were merge-sorted, and
 where merge-sort was off and no droplets were merge-sorted. For all images
 in this folder, ***X3*** is *ch00* for the brightfield image channel,
 *ch01* for Calcein-green fluorescence channel, *ch02* for Cell Trace
 Yellow fluorescence channel, and *ch03* for Cell Trace Far-red
 fluorescence channel. Images taken at 10X magnification include *10X* in
 the file name, and images taken at 4X and stitched together include
 *4X_Stitched* in the file name. These images collectively were used
 generate figures 3 and S6.  ##### Folder: Merge-sort_All
 **Description:** Contains fluorescent microscopy images when every droplet
 pair is merge-sorted. The files *Merge-sort_All_10X_**X3**.jpg* were used
 to generate the figure image, and the
 files *Merge-sort_All_4X_Stitched_**X3**.jpg* were analyzed to determined
 cell encapsulation results. An example file name is
 *Merge-sort_All_10X_1_ch01.jpg*, which is droplets collected from the
 merge-sort all condition at a 10X magnification in the calcein-green
 channel.  ##### Folder: Merge-sort_Cell1&2 **Description:** Contains
 fluorescent microscopy images when only drops containing cell 1 and 2 are
 merged. The files *Merge-sort_Cell1&2_10X_**X3**.jpg* were used to
 generate the figure image, and the
 files *Merge-sort_Cell1&2_4X_Stitched_**X3**.jpg* were analyzed to
 determined cell encapsulation results. An example file name
 is *Merge-sort_Cell1&2_4X_Stitched_2_ch01.jpg*, which is a stitched,
 4X magnification image of drops collected from the cell 1 and 2 merge-sort
 condition in the calcein-green channel.  #####
 Folder: Merge-sort_Cell1&3 **Description:** Contains fluorescent
 microscopy images when only drops containing cell 1 and 3 are merged. The
 files *Merge-sort_Cell1&3_10X_**X3**.jpg* were used to generate the
 figure image, and the
 files *Merge-sort_Cell1&3_4X_Stitched_**X3**.jpg* were analyzed to
 determined cell encapsulation results. An example file name
 is *Merge-sort_Cell1&3_4X_Stitched_2_ch01.jpg*, which is a stitched,
 4X magnification image of drops collected from the cell 1 and 3 merge-sort
 condition in the calcein-green channel.  ##### Folder: Merge-sort_Off
 **Description:** Contains fluorescent microscopy images when no drops are
 merged. The file *Merge-sort_Off_10X_3_**X3**.jpg* was used to generate
 the figure image, and the
 files *Merge-sort_Off_10X_1_**X3**.jpg*, *Merge-sort_Off_10X_2_**X3**.jpg*, and *Merge-sort_Off_10X_3_**X3**.jpg* were analyzed to determined cell encapsulation results. An example file name is *Merge-sort_Off_10X_1_ch01.jpg*, which is a 10X magnification image of drops collected from the merge-sort off condition in the calcein-green channel.  #### Folder: Fig4+FigS7 **Description:** Data supporting figures 4 and S7 of the manuscript. Contains folders *K562_Starting_Calcein_Green, Merge_All, Merge-sort*, and *Selective_Merger* which respectively contain fluorescent microscopy images (*.jpg*) of K562 cell stained with calcein-green before microfluidic encapsulation, collected alginate beads when all drops were merged, collected alginate beads when only cell pair drops were merge-sorted, and collected aggregated alginate gel when only selective merger is performed. **X4** is *ch00* for the bright field image channel, *ch01* for Cell Trace Far-red fluorescence channel, *ch02* for Cell Trace Yellow fluorescence channel, and *ch03* for Calcein-green fluorescence channel. Images taken at 10X magnification include *10X* in the file name, and images taken at 4X and stitched together include *4X_Stitched* in the file name. Images in the *Merge_All* and *Merge-sort* folders were used to measure alginate bead size, frequency of cell pairings in alginate beads, and viability of cells in alginate beads at time points of 0 and 18 hours after encapsulation.  ##### Folder: K562_Starting_Calcein_Green **Description:** Contains fluorescent microscopy images of K562 cells stained with calcein green before entering a microfluidic workflow. The files *K562_starting_calcein_green_10X_**X4**.jpg* were analyzed to determine frequency of calcein-green positive cells. An example file name is *K562_starting_calcein_green_10X_1_ch00.jpg*, which is a 10X magnification image of the bright field channel for sample 1 used to determine starting viability of the K562 cells.  ##### Folder: Merge_All **Description:** Contains fluorescent microscopy images of stained K562 cells in alginate beads when all droplet pairs are merged and collected. The files *merge_all_0hrs_10X_**X4*** were used the generate the figure image. Files *merge_all_0hrs_4X_Stitched_**X4*** and *merge_all_18hrs_4X_Stitched_**X4*** were used to determine bead diameter, frequency of cell pairings, and viability and timepoints 0 and 18 hours. An example file name is *merge_all_18hrs_4X_Stitched_ch03.jpg*, which is a stitched, 4X magnification image, of the merge all cells in alginate beads in the calcein-green channel, 18 hours after droplet breaking.  ##### Folder: Merge-sort **Description:** Contains fluorescent microscopy images of stained K562 cells in alginate beads when only the correct cell pair droplets are merged and collected. The files *merge_sort_0hrs_10X_**X4*** was used the generate the figure image. Files *merge_sort_0hrs_4X_Stitched_**X4*** and *merge_sort_18hrs_4X_Stitched_**X4*** were used to determine bead diameter, frequency of cell pairings, and viability and timepoints 0 and 18 hours. An example file name is *merge_sort_18hrs_4X_Stitched_ch03.jpg*, which is a stitched, 4X magnification image, of the merge-sorted cells in alginate beads in the calcein-green channel, 18 hours after droplet breaking.  ##### Folder: Selective_Merger **Description:** Contains fluorescent microscopy images of stained K562 cells in alginate aggregate when cell pair droplets are selectively merged only and not sorted. The files *selective_merger_0hrs_10X_**X4*** were used the generate the figure image. An example file name is *selective_merger_0hrs_10X_ch00.jpg*, which is a 10X magnification image in the bright field channel of the selective merger condition cells in alginate right after droplet breaking.  #### Folder: FigS1 **Description:** Data supporting figure S1 of the manuscript. Contains 10X magnification, bright field microscopy images (.tif) of paired droplets at aqueous flow rates of 220 uL/hr to 500 uL/hr for the big droplet using constant oil flow rates. The files *figS1_10X_**XS1*** where **XS1** is the aqueous flow rate in uL/hr for each condition, were used to generate the figure and calculate distance between successive droplets. An example file name is *figS1_10X_220.tif,* which is a 10X magnification image of the paired droplets when the aqueous flow rate generating the big droplet is 220 uL/hr.  #### Folder: FigS2 **Description:** Data supporting figure S2 of the manuscript. Contains 10X magnification, bright field microscopy images (.tif) of droplet pairs with different big droplet diameters used to generate figure. The files used for the figure are named *figS2_10X_**XS2***, where **XS2** is the diameter in microns of the big droplet in that image. The decimal point is represented by a "-," for instance 71.1 is represented by 71-1 in the filename. An example file name is *figS2_10X_71-1.tif*, which is a 10X magnification image of a droplet pairing when the big drop has a 71.1 um diameter.  #### Folder: FigS3 **Description:** Data supporting figure S3 of the manuscript. Contains folders *Collection* and *Snapshots*, which contains images of the collection at different event rates and snapshots of the merge-sort area during stable, transition, and unstable merge-sorting event rates.  ##### Folder: Collection **Description:** Contains images (.jpg) of the collected merge-sorted droplets at varying event rates from 105 to 929 events per second. The files are named *ER_XS3-1_4X_XS3-2_XS3-3*, where **XS3-1** is the event rate in events/second, **XS3-2** is the sample number, and **XS3-3** is *ch00* for the bright field channel, *ch01* for the BSA-Cy5 fluorescence channel, and *ch02* is the FITC fluorescence channel. Example file name is *ER_105_4X_1_ch00.jpg*, where the image is for an event rate of 105 events/s, sample 1, and the bright field channel. These images were used to calculate correct merge-sort percentages. ##### Folder: Snapshots **Description:** Contains snapshots (.tif) of the merge-sort area during the trigger point, when droplets merge, when the merged droplet is sorted, and when the trailing drop flows into the waste. The files are named ***XS3-4_XS3-5_XS3-6***, where **XS3-4** is the regime state: *stable*, *transition*, or *unstable*, **XS3-5** is the snapshot number, and **XS3-6** is the description of the snapshot: *trigger_point* for the trigger point, *merge* for when the droplets merge, *sort* for when the merged drop is sorted, and *trailing_drop* for when the trailing drop flows into the waste. Example file name is *stable_1_trigger_point.tif*, where the image is the snapshot of the trigger point for merge-sorting during the stable regime.  #### Folder: FigS4 **Description:** Data supporting figure S4 of the manuscript. Excel spreadsheet figS4_PMT_time+voltages.xlsx contains PMT voltages (volts) and times (ms) for paired fluorescent droplets under the 4X, 10X, 20X, and 40X objectives examined in figure S4. Images (.tif) figS4_**XS4**, where **XS4** is the magnification of the objective were used to generate the figure. Example file name is *figS4_4X.tif*, where the image is a view of the merge-sort area using the 4X objective.  #### Folder: FigS5 **Description:** Data supporting figure S5 of the manuscript. Contains folders *drops_in_sort_area*, *merge-sort_all*, *merge-sort_off*, and *merge-sort_peak_duration_gate*, and the file *figS5_PMT_Voltage+Time.xlsx,* which contains PMT voltages (volts) and times (ms) of the droplets examined in figure S5.  ##### Folder: drops_in_sort_area **Description:** Contains snapshots (.tif) when a larger drop is paired with the big drop (drops_in_sort_area_large_paired_drop), when a small drop is paired with the big drop (drops_in_sort_area_small_paired_drop), and when there is only a big drop (drops_in_sort_area_no_paired_drop).  ##### Folder: merge-sort_all **Description:** Contains images (.jpg) when all drops were merge-sorted regardless of peak duration. The files are named merge-sort_all_**XS5-1**, where **XS5-1** is *ch00* for the bright field channel, *ch01* for the BSA-Cy5 fluorescence channel, *ch02* for the rhodamine B fluorescence channel, and *ch03* for the FITC fluorescence channel. Example file name is *merge-sort_all_ch02.jpg*, which is an image of the merge-sort all collected droplets in the rhodamine B fluorescence channel.  ##### Folder: merge-sort_off **Description:** Contains images (.jpg) of the waste when merge-sorting is turned off. The files are named merge-sort_off_**XS5-2**, where **XS5-2** is *ch00* for the bright field channel, *ch01* for the BSA-Cy5 fluorescence channel, *ch02* for the rhodamine B fluorescence channel, and *ch03* for the FITC fluorescence channel. Example file name is *merge-sort_off_ch02.jpg*, which is an image of the merge-sort off collected waste droplets in the rhodamine B fluorescence channel.  ##### Folder: merge-sort_peak_duration_gate **Description:** Contains images (.jpg) when drops were merge-sorted on a peak duration gate to only select droplet pairs with the small input droplet. The files are named merge-sort_peak_duration_gate_**XS5-3**, where **XS5-3** is *ch00* for the bright field channel, *ch01* for the BSA-Cy5 fluorescence channel, *ch02* for the rhodamine B fluorescence channel, and *ch03* for the FITC fluorescence channel. Example file name is *merge-sort_peak_duration_gate_ch02.jpg*, which is an image of the peak duration gated merge-sort collected droplets in the rhodamine B fluorescence channel.