Robust Double\nEmulsions for Multicolor Fluorescence-Activated\nCell Sorting

Cell–cell interactions are essential for the proper\nfunctioning\nof multicellular organisms. For example, T cells interact with antigen-presenting\ncells (APCs) through specific T-cell receptor (TCR)–antigen\ninteractions during an immune response. Fluorescence-activated droplet\nsorting (FADS) is a high-throughput technique for efficiently screening\ncellular interaction events. Unfortunately, current droplet sorting\ninstruments have significant limitations, most notably related to\nanalytical throughput and complex operation. In contrast, commercial\nfluorescence-activated cell sorters offer superior speed, sensitivity,\nand multiplexing capabilities, although their use as droplet sorters\nis poorly defined and underutilized. Herein, we present a universally\napplicable and simple-to-implement workflow for generating double\nemulsions and performing multicolor cell sorting using a commercial\nFACS instrument. This workflow achieves a double emulsion detection\nrate exceeding 90%, enabling multicellular encapsulation and high-throughput\nimmune cell activation sorting for the first time. We anticipate that\nthe presented droplet sorting strategy will benefit cell biology laboratories\nby providing access to an advanced microfluidic toolbox with minimal\neffort and cost investment.