preparation of functionalized magnetic nanoparticles for time-resolved fluorescent imaging

Microfluidic Droplet Assay for Single-Cell Cytokine Secretion Analysis

Infections often cause complex host reactions involving the innate and adaptive immune systems1,2. Upon infection or recognition of infectious agents, host cells can produce a diverse range of chemo- and cytokines, which are small proteins known as critical communicators and modulatory of the immune system3. Pro-inflammatory cytokines are released early upon infection to initiate the immune response, followed later on by anti-inflammatory cytokines, which are critical to prevent tissue damage and subsequent chronic or autoinflammatory diseases. This balance between threat elimination and tissue protection manifests as a wide repertoire of cytokines exerting different functions during the infection, allowing for a fine-tuning of the response4,5. Within this mixture, unique signatures can be observed depending on the pathogen and the signals they induce, the tissue location, and the immune cells from which they originate. However, cytokine release also appears to constitute a multi-functional biological process unique to each cell population, diverse in secretion dynamics and individual response. This heterogeneity has been described in the literature for many years, for instance, among T-cell subpopulations6,7, where investigations into autoinflammatory diseases and severe COVID-19 infections exhibited a large functional diversity of inflammatory markers within and in between patients8,9. Lately, the advent of single-cell sequencing highlighted the high plasticity and crosstalk between subpopulations within immune microenvironments that were not previously apparent, indicating that single-cell methods are necessary to capture this heterogeneity10,11. While novel methods are being developed to analyze the transcriptome, phenotypic analysis remains challenging, as this requires simultaneous, quantitative, and time-resolved measurements of protein secretion on a single-cell level. Such measurements allow us to investigate secreting cell identities, dynamics, and secretion patterns (slow/fast, early/late, simultaneous/sequential) for a repertoire or a panel of cytokines. By enabling the study of the dynamics of cytokine release during an immune response quantitatively and with temporal resolution, the resulting insights might allow for an understanding of the cellular ensemble and the induced response.
In standard protocols, cytokines are usually detected in the supernatant of cell suspensions and serum using enzyme-linked immunosorbent assay (ELISA), yielding bulk-secreted amounts. Bulk measurements do not allow for quantification of the cytokine amounts produced by each cell, a problem especially highlighted in heterogeneous cell samples. Alternative methods such as intracellular cytokine staining, enzyme-linked immunospot (ELISpot) assay or micro-engraved assays (e.g., Isoplexis) detect cytokines expressed by individual cells but provide endpoint measurements only12,13. This means that secretion dynamics and changes that might happen in the cellular secretion pattern over the incubation time are ignored. Additionally, endpoint measurements cannot differentiate between simultaneous and sequential cytokine secretion, so the true extent of simultaneous polyfunctionality of immune cells in cytokine secretion remains unclear using these methods.
Single-cell resolution can be achieved using droplet microfluidics to generate and process picoliter-sized physical compartments in order to study immune cells on their unique cytokine secretion phenotypes on the single-cell level14,15. These compartments consist of water-in-oil emulsions and can be generated using microfluidic chips16,17. Indeed, droplet-based microfluidic assays have demonstrated extreme versatility in enabling the analysis of different biological samples and repertoires on the single-cell level and their integration with upstream (cell and reagent processing) and downstream processes (single-cell sorting, proteomics or sequencing)18,19,20,21,22. In particular, setups that permit droplet immobilization allow for the measurement of a single-cell functionality over time, which is valuable for the analysis of protein secretion18. Furthermore, integrating multiplexed, quantitative assays facilitates additional investigations in previously inaccessible dimensions, into processes such as co-secretion and the identification of polyfunctional immune cells23,24.
In this protocol, we describe an immobilized droplet-based single-cell workflow to detect, quantify and temporally measure the secretion of up to three cytokines in parallel from individual cells17,23. The technology offers the ability to monitor cytokine responses from over 20,000 cells in parallel.
The presented workflow consists of the microfluidic encapsulation of single immune cells and functionalized nanoparticles into 60 pL water-in-oil droplets. The immobilization of &#62;100,000 droplets in an observation chamber and time-resolved fluorescence microscopy allow for the measurement of cytokine secretion dynamics within each droplet and each cytokine (Figure 1A). For each individual cell within a droplet, the cytokine secretion is measured by a sandwich immunoassay, where magnetic nanoparticles functionalized with a specific capture antibody bind the secreted cytokine, leading to the subsequent relocation and binding of fluorescently labeled detection antibodies (Figure 1B,C). A beadline is formed by aligning the magnetic nanoparticles, to which fluorescence relocation can be quantified in the presence of cytokine. Here, fluorescence relocation is defined as the average fluorescence intensity found on the beadline divided by the average fluorescence intensity of the remaining droplet. This assay can be multiplexed for several cytokines by mixing differently functionalized nanoparticle batches and respective detection antibodies labeled in different fluorescence channels23, resulting in specific fluorescence relocations in the different channels. With the help of a customized analysis script, fluorescence relocation values can be extracted, and the images can be converted into secretion dynamic profiles for every individual cell and cytokine. Therefore, the resulting datasets yield numerous readouts, such as the quantitative secretion measurement over time, the identification of co-secreting subpopulations, and the distributions of the cells according to secreted amounts, rates, and combinations of cytokines.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66492/66492fig01.jpg" /> 
Figure 1: Workflow and assay principle. (A) Overview of the workflow for analyzing&#160;cytokine-secreting cells after stimulation. Single-cell suspensions and magnetic nanoparticles are prepared and encapsulated into 60 pL in volume oil/water emulsions (droplets). Droplets are immobilized and nanoparticles aligned inside a magnetic field before measurement for up to 4 h every 30 min. Finally, images are analyzed, and the parameters for every droplet, timepoint and fluorescent channel are extracted. This figure has been modified from17. (B) Principle of the droplet sandwich bioassay. Functionalized nanoparticles bind the secreted cytokines, which leads to the subsequent relocation of fluorescently labeled detection antibodies to the nanoparticles. This relocation of fluorescence is quantified and validated with calibration experiments performed with recombinant cytokines. Mixing different functionalized nanoparticles allows the multiplexed measurements of up to three cytokines simultaneously. (C) In cell-based experiments, droplets are followed over the measurement time and secreting cells are identified by an overtime increase of fluorescence relocation onto the nanoparticles. Schematics are not up to scale. Figure created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/66492/66492fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Unveiling the Polyfunctionality and Heterogeneity in Immune Responses

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells 

In this project, we focus on developing and applying novel analytical strategies, which allow for the direct, quantitative, and deep analysis of cellular functionalities at single-cell resolution. And so by going beyond pure one data point measurement, we aim to better understand and exploit this multi-functionality, which is coming from complex responses in the host and in tissue. To obtain a comprehensive understanding of these functionalities is complex, difficult, and requires interdisciplinary approaches.And so one challenge in this context is to ensure that the dynamic range of the assay develop is appropriate to capture the quantity and dynamics of a secreted protein, which can vary greatly across samples, preparations, and tissues. Disruption in the regulation of the immune response can induce various disorder, leading to mild fever to potentially life-threatening complication. Moving beyond traditional endpoint measurements, we proposed to directly measure the dysregulation and activation of immune cells dynamically on a single-cell level to gain additional insight.So cytokine are usually detected at the specific time point as a concentration in the supernatant or concentration of activated cells. Bulk measurement directly to quantify secretion of each individual cells, masking cellular heterogeneity who, as endpoint measurements, do not distinguish between simultaneous and secondary secretion. Overlooking the dynamic aspect of the response Cellular fully functionality refers to an immune cell's ability to secrete multiple cytokines simultaneously.This allows them to fine tune their response against threats, for instance, and to measure this poly functionality dynamically, meaning over time can give an over insights into the different nuances of an immune response.

Preparation of Functionalized Magnetic Nanoparticles for Time-Resolved Fluorescent Imaging

To begin, add streptavidin functionalized nanoparticles to tubes for TNF alpha, interleukin-1 beta, and interleukin-6 detection. Dilute the streptavidin plus nanoparticles in equal volumes of PBS. Then, add the biotinylated capture antibody solutions to tubes and incubate for 30 minutes at room temperature.Next, add 0.01%of 1 millimolar D-Biotin solution to each tube and incubate for five minutes. Then, hold a neodymium magnet close to the tube to collect the particles. Discard the clear supernatant and immediately resuspend the nanoparticles in a mixture of Pluronic F-127 and PBS and incubate for 30 minutes.After incubating the particles for 30 minutes in storage buffer, stir them to ensure a uniform mixture. Immediately before encapsulation, combine the conjugated nanoparticles for interleukin-6, TNF alpha, and interleukin-1 beta. Now, wash the particles with complete media using the magnet as demonstrated earlier, and resuspend the beads in complete media.Afterward, add detection antibodies for interleukin-6, TNF alpha, and interleukin-1 beta to achieve the final concentration of 10 nanomolar.

Research

JoVE Journal

Immunology and Infection

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. ...