Multiplexed profiling of single-cell secretion on a hierarchical loading microwell chip.

Protein secretion is involved in many biological processes, such as cellular communication, embryonic development, tissue homeostasis, disease pathogenesis, and execution of immune functions. Highly multiplexed detection of single-cell secretion is greatly needed but has always been challenging. Herein, we present a hierarchical loading microwell chip (HL-Chip) -based method that efficiently aligns thousands of single cells with multiple antibody-coated microbeads for highly multiplexed detection of secreted proteins from single cells. We demonstrate the applications of this platform in profiling secretion of six cytokines from single T cells and macrophages after pan- and antigen-specific stimulation. The 6-plex cytokine profiling reveals an early but transient cytokine burst and polyfunctional heterogeneity in antigen peptide-stimulated T cell receptor-engineered T (TCR-T) cells. This simple and versatile technology could find its application in single-cell measurements in both biological discoveries and clinical diagnosis.