Data from: Deterministic cell pairing with simultaneous microfluidic merging and sorting of droplets
Data files
Oct 18, 2025 version files 1.42 GB
-
data_from_Joslin_et_al_2025.zip
1.42 GB
-
README.md
15.47 KB
Abstract
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 < 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, > 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.
Dataset DOI: 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.
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.