Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving

Abstract

Summary The large diversity of cells that comprise the human immune system requires methods that can resolve the individual contributions of specific subsets to an immunological response. Microengraving is process that uses a dense, elastomeric array of microwells to generate microarrays of proteins secreted from large numbers of individual live cells (~104–105 cells/assay). In this paper, we describe an approach based on this technology to quantify the rates of secretion from single immune cells. Numerical simulations of the microengraving process indicated an operating regime between 30 min-4 h that permits quantitative analysis of the rates of secretion. Through experimental validation, we demonstrate that microengraving can provide quantitative measurements of both the frequencies and the distribution in rates of secretion for up to four cytokines simultaneously released from individual viable primary immune cells. The experimental limits of detection ranged from 0.5 to 4 molecules/s for IL-6, IL-17, IFNγ, IL-2, and TNFα. These multidimensional measures resolve the number and intensities of responses by cells exposed to stimuli with greater sensitivity than single-parameter assays for cytokine release. We show that cells from different donors exhibit distinct responses based on both the frequency and magnitude of cytokine secretion when stimulated under different activating conditions. Primary T cells with specific profiles of secretion can also be recovered after microengraving for subsequent expansion in vitro. These examples demonstrate the utility of quantitative, multidimensional profiles of single cells for analyzing the diversity and dynamics of immune responses in vitro and for identifying rare cells from clinical samples.

A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis

“…After a short incubation (1-2 hours), the glass is removed to yield an array of cytokines registered to the array of microwells. Using this method, we have demonstrated, and confirmed with quantitative models, the ability to measure the secretion of IFN-γ, IL-2, and TNF-α simultaneously from polyclonally activated, primary T cells ex vivo with sensitivities 10- to 100-fold greater than ELISpot and surface-based capture (27).…”

“…In the absence of a direct assay to measure both functions (cytolysis and secretion), it has remained unclear to what extent cells exhibit both responses concurrently when encountering an APC. We have previously shown that the elastomeric array of microwells used here can also detect the frequencies and rates of secretion of multiple cytokines by a process called microengraving (26,27). According to this method, a glass slide coated with antibodies specific for .…”

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

“…In addition, because the droplets in the above methods are fully sheathed by a carrier fluid, they are designed primarily for studies with suspended cells. There are methods that bypass the production and capturing under flow altogether by initially producing stationary plugs within the geometry of the device (2,(23)(24)(25). However, upon their adjustment to prolonged mammalian assays, they require multilayer fabrication, either involving high-pressure cell loading or not allowing for different substrates to be used (26,27).…”

Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

“…However, the limited protein detection range and the lack of detection sensitivity of current techniques are obstacles in the monitoring and study of secreted proteins from single cells. 3,4 One of the ways to circumvent such concerns is the use of protein microarrays that are commonly utilized for the high-throughput screening and analysis of protein interactions. [5][6][7] Protein microarrays rely on the capture of secreted proteins from cells on a support surface, for example, a nitrocellulose membrane, glass slide, or microtiter plate, and detection by the fluorescent dye-labeled probe molecules added to the array.…”