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.

preparation-functionalized-magnetic-nanoparticles-for-time-resolved

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. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

The protocol describes an advanced microfluidic platform to quantitatively measure cytokine secretion dynamics of individual human peripheral blood mononuclear cells. The platform measures up to three cytokines in parallel (IL-6, TNF&#945;, and IL-1&#946;) for each individual cell stimulated with lipopolysaccharide as an example.

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

Observation Chamber Fabrication for Imaging Single-Cell Suspensions and Magnetic Nanoparticles

To begin, place the glass slide with holes on a clean surface with the plasma-activated side facing upwards. Carefully remove the protective layer from one side of the double-sided adhesive tape in the direction of cutting. Align the tape with the edges of the glass slide and the drilled holes.Place the tape on the glass slide starting from the short edge and make sure of good adherence. After removing the second protective layer from the tape, place the second glass slide with the activated surface facing downwards. Using a flat board, press the two glass slides together while applying the upper body strength for 10 seconds.Flip the chamber so that the holes face the user. Apply a small amount of UV curable glue in the ring below the NanoPort. Slide the NanoPort down the hole in the glass slide using a microtubing and add a ring of UV curable glue around the port.Load one milliliter of 1%fluorosilane solution prepared in fluorinated oil into a syringe. Inject the solution through a PTFE syringe filter and a needle attached to PTFE microtubing into the observation chamber. After a minute of incubation, use nitrogen pressure to flush out the coating solution under a fume hood.Using a different syringe assembly, rinse the chamber solely with fluorinated oil.

Encapsulation of the Cells and Nanoparticles into Droplets for Fluorescent Imaging

To begin, fill a one milliliter syringe with 500 microliters of continuous phase. Attach the PTFE microtubing to a 27 gauge 0.75 inch needle. Mount the assembly onto the syringe and subsequently onto the syringe pump.Then punch a hole using a 0.75 millimeter biopsy punch in the middle of a PDMS cutout. Pull approximately three centimeters of PTFE tubing through the hole in the cutout, and push the assembly into the top of a 200 microliter pipette tip. Seal the connector with UV curable glue on top of the pipette, and attach the other side of the tube to a 23 gauge 1.25 inch needle.Next, fill two one milliliter syringes with 500 microliters of light mineral oil. Attach two 23 gauge needles with the custom-made attachments and mount them onto the syringe pump. Using the syringe pump control software, aspirate functionalized nanoparticle and cell solution into the pipette tip of the aqueous phases.After cleaning the prepared observation chamber, clamp the chamber into the printed chamber holder equipped with two neodymium magnets at 30 degrees. Now, open both ports and plug a paper towel in the upper port to absorb the excess outer phase during filling. Connect the continuous phase to the top inlet of the microfluidic chip.Then, flush the chip with the continuous phase for approximately 30 seconds at a flow rate of 1800 microliters per hour. Attach the pipette tips of the aqueous solutions to the two middle inlets of the chip. Start the flow of the aqueous solution at 200 microliters per hour for each solution.Once the liquid appears at the outlet, begin the fluorinated oil phase flow at 800 microliters per hour. Wait until stable droplet production is established, indicated by a homogeneous gray, shiny solution at the outlet. Then connect the PTFE microtubing to the outlet port to collect the droplets.Using the ferrule module of a finger-tight one piece fitting, direct them into an observation chamber. Once the chamber is filled, stop the flow and close off the ports with port plugs using finger-tight pressure. After droplet production, flush the chip with fluorinated oil, followed by nitrogen, to blow out any remaining liquid.Mount the chamber holder on the microscope with a well plate format stage. Then, using the 10x objective, switch the microscope to the brightfield channel. Focus on the immobilized droplets in the brightfield, and move to the center of the chamber.Then, activate the automated focusing system. Adjust the measurement plane on the brightfield channel so that droplet edges appear as sharp black circles easily distinguishable from the oil phase and background. Go through each of the fluorescence channels, setting the optimal measurement plane for them.For relocation measurements, focus on the nanoparticle aggregate in the FITC, TRITC, and Cyanine5 channels.