PDMS Microwell Stencil Based Multiplexed Single‐Cell Secretion Analysis

Multiplexed single-cell protein secretion analysis provides an in-depth understanding of cellular heterogeneity in intercellular communications mediated by secreted proteins in both fundamental and clinical research. However, it has been challenging to increase the proteomic parameters co-profiled from every single cell in a facile way. Herein, a simple method to improve the multiplexed proteomic parameters of PDMS microwell based single-cell secretion analysis platform by sandwiching PDMS stencil in between two antibody-coated glass slides is introduced. Two different antibody panels can be immobilized easily by static coating, without using sophisticated fluid handling or bulky equipment. 5-plexed, 3-fluorescence color single-cell secretion assay is demonstrated with this platform to investigate human monocytic U937 cells in response to lipopolysaccharide and phorbol myristate acetate stimulation, which identified the existence of functional subsets dictated by different cytokine profiles. The technology introduced here is simple, easy to operate, which holds great potential to become a powerful tool for profiling multiplexed single-cell cytokine secretion at high throughput to dissect cellular heterogeneity in secretome signatures.