Deterministic cell pairing with simultaneous microfluidic merging and sorting of droplets

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..., 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.
, # 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_J...,