A Guide to the Quantitation of Protein Secretion Dynamics at the Single-Cell Level.

Protein secretion is a key cellular functionality, particularly in immunology, where cells can display large heterogeneity in this crucial activity in addition to binary secretion behavior. However, few methods enable quantitative secretion rate measurements at the single-cell level, and these methods are mostly based on microfluidics systems. Here, we describe such a microfluidic single-cell method for precisely measuring protein secretion rates in detail, building on the published droplet-based microfluidic platform DropMap. We give an updated, detailed guide toward quantifying protein secretion rates, discussing its setup and limitations. We illustrate the protocol on two key immunological analytes, immunoglobulin G, and interferon-γ.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.