| Abstract | Cell signaling is modulated by the secretion of various proteins, which can be used to infer a cell's phenotype. However, these proteins cannot be readily detected in multiplex by commonly used methods at the single-cell level. Here we present PiP-plex a particles-in-particle (PiPs) system for multiplex protein secretion analysis by confocal microscopy. PiP-plex-comprises (i) fluorescence intensity barcoded microparticles (BMPs) co-entrapped with (ii) a single cell inside an alginate hydrogel particle. We found that PiPs maintained >90% cellular viability and allowed live cells retrieval. A seven-plex fluorescent barcoding and concomitant sandwich immunoassay in PiPs was developed with limits of detection ranging from 0.8 pg mL⁻¹ to 2 ng mL⁻¹ depending on the protein. PiP-plex assays were benchmarked with bulk immunoassays and found to rival or outperform them. Proteins secreted by single THP-1 cells upon exposure to lipopolysaccharide were measured by PiP-plex and varying cell responses detected, including a significant increase in MIP-1α, TNF-α, and IL-17A; MIP-1α and IL-17A were the most frequently secreted cytokines, while other cytokines were typically co-secreted. Using PiP-plex, we analyzed ≈750 THP-1 cells, showcasing its potential for characterizing cells and cell-based therapeutics for e.g. cancer immunotherapies. |
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