ASR designed experiments, developed protocols and analyzed data. SC and PS performed cell size experiments. ASR and OC wrote the manuscript. The authors wish to gratefully acknowledge the support of Prof. David R. Klug for providing access to equipment. The authors wish to thank the Imperial College Advanced Hackspace for access to fabrication and prototyping facilities.

<ol>
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The authors have nothing to disclose.

Addressable Droplet Microarrays are a sensitive and extensible method for quantitatively determining the absolute copy number of protein within a single cell.
Limiting the level of non-specific binding (NSB) is critical within the protocol to achieving as low a limit of detection as possible. Proteins and other biochemical species may non-specifically bind to a number of interfaces present within the droplets &#8212; the coverslip surface, the antibody spot and the oil/water interface. Protein...

The goal of this method is to determine the variation in abundance of a target protein in a cell population with single cell resolution. Single cell analysis provides a number of benefits that are not available with traditional ensemble biochemical methods.1,2,3,4,5 Firstly, working at the single cell level can capture the rich heterogeneity of a cell population that would otherwise be lost by the averaging that occurs with traditional ensemble biochemical techniques. The majority of work-horse biochemical methods work with the bulk, requiring, as they often do, millions of cells to produce a result. Of course, the consequences of assessing entire cell populations depends on a number of factors, for example, the heterogeneity in protein expression where some important features of the distribution of protein abundance may be missed. From a practical perspective, the sensitivity required of single cell techniques make them capable of working with amounts of biological material that is insufficient for even the more sensitive bulk techniques to function. A key example of this is the study of rare cell types such as circulating tumor cells (CTCs) where even for patients with a poor prognostic outlook less than 10 CTCs might be present in a single 7.5 mL blood draw.6 Here we present the methodology required to perform single cell protein measurements using a reduced volume antibody-based assay employing oil-capped droplets printed on an antibody microarray.
Microfluidic droplet platforms are high throughput, able to generate thousands of droplets per second, and capable of isolating, and even culturing, single cells in individual droplets to perform a wide array of biochemical assays. Droplet-based techniques are well suited for single cell analysis,7,8,9 with notable recent examples including DropSeq10 and inDrop11, which have been greatly aided by the power of amplification techniques. The limited amount of material and no methods of amplification for proteins make single cell proteomics especially challenging.
Droplets may be analyzed by a number of methods and fluorescence microscopy has been widely used. Single molecule techniques such as total internal reflection fluorescence (TIRF) microscopy allows fluorescent molecules to be visualized with unparalleled signal-to-noise ratio.12 Due to the exponential decay of the evanescent field, only fluorophores in high proximity to the surface (order of 100nm) are excited making TIRF a good strategy in detecting small amounts of a target molecule in a complex mixture. The inherent optical sectioning strength of TIRF also helps to avoid wash steps and limits assay time and complexity. However, TIRF requires planar surfaces and examples of TIRF microscopy applied to droplets in flow involve the formation of a planar surface of which to image.13 To this end, single cell proteomic techniques often design microfluidic chips around surface-immobilized capture agents in a microarray format.4,14
The droplets, themselves, may be formed in arrays on planar surfaces, so-called droplet microarrays.15,16,17 Spatially organizing droplets into arrays allows them to be conveniently indexed, easily monitored over time, individually addressed and, if required, retrieved. Droplet microarrays can achieve a high density of micro-reactors with thousands of elements per chip which are either free-standing or supported by microwell structures.18,19,20 They may be formed by sequential deposition by liquid handling robots, inkjet spotters, contact microarrayers21,22,23,24,25,26 or they can self-assemble on surfaces such as superhydrophillic spots patterned on a superhydrophobic surface.27,28,29
With these considerations in mind, Addressable Droplet Microarrays (ADMs) were designed to combine the versatility, spatial addressability and reduced volumes of droplet microarrays with the sensitivity of single molecule TIRF microscopy to quantitatively measure protein abundance.5 ADMs enable single cell analysis forming a droplet microarray containing single cells over an antibody microarray, which is then capped with oil to prevent evaporation. The volumes of the droplets are discrete to prevent sample loss, which would otherwise be achieved by on-chip valving in continuous flow microfluidics.30 The absolute amount of target protein from a single cell is extremely small; however, the reduced volume of the droplets allows for relatively high local concentration in order that they are detected using a sandwich antibody assay - antibody is immobilized in a distinct region, or spot, on a surface which captures protein which in turn binds to a fluorescently labelled detection antibody present in the droplet volume. As a label-free approach (i.e. protein targets do not need to be labelled directly), ADMs are generally applicable to analyzing cells from primary sources, such as processed blood, fine need aspirates and dissociated tumor biopsies, as well as cells from culture and their lysates.
Measuring the variation in protein abundance across a cell population is important in determining the heterogeneity in response, for example, to a drug and will help in providing insight into cellular functions and pathways, assessing subpopulations and their behavior as well as identify rare events that would otherwise be masked by bulk methods. This protocol describes how to produce and use addressable droplet microarrays to quantitatively determine the abundance of the transcription factor p53 in human cancer cells and may be used to investigate the role of p53 in response to chemotherapeutic drugs. The target protein is determined by the choice of capture and detection antibodies and may be modified to include more or different targets. Instructions are provided to build a simple apparatus incorporating a concentric nozzle from general lab consumables to manually array 10 nL droplets capped with oil. The full experimental process is described whereby each droplet is then loaded with a single cell, which is then lysed and the expression of protein determined with single molecule resolution using TIRF microscopy.

<table><tbody><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Life Technologies</td><td>10010015</td><td></td></tr><tr><td>DMEM high glucose</td><td>Sigma</td><td>D6429</td><td></td></tr><tr><td>Foetal Bovine Serum (FBS)</td><td>Biochrom</td><td>S0115</td><td></td></tr><tr><td>cell culture flasks</td><td>Corning</td><td>SIAL0639</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Biochrom</td><td>L2153</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microarray&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microcontact Arrayer</td><td>DigiLab, UK</td><td>OmniGrid Micro</td><td></td></tr><tr><td>Microcontact pin</td><td>ArrayIt, USA</td><td>946MP2</td><td></td></tr><tr><td>Coverslips (Nexterion)</td><td>Schott, Europe</td><td>1098523</td><td>Size (mm): 65.0 x 25.0; Thickness (mm) 0.17</td></tr><tr><td>p53 capture antibody</td><td>Enzo</td><td>ADI-960-070</td><td></td></tr><tr><td>p53 detection antibody, Alexa Fluor 488 labelled</td><td>Santa Cruz</td><td>sc-126</td><td>stock concentration 200&amp;mu;g/mL</td></tr><tr><td>Saline-sodium citrate buffer</td><td>Gibco</td><td>15557-044</td><td></td></tr><tr><td>Betaine</td><td>Sigma</td><td>61962</td><td></td></tr><tr><td>Sodium dodecyl sulphate</td><td>Sigma</td><td>L3771</td><td></td></tr><tr><td>384 well plate (low volume)</td><td>Sigma</td><td>CLS4511</td><td></td></tr><tr><td>Nitrogen gas cylinder</td><td>BOC</td><td>Industrial grade, oxygen-free</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Droplets&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>Eppendorf</td><td>Patchman NP2</td><td></td></tr><tr><td>Manual Microinjector</td><td>Eppendorf</td><td>CellTram Vario</td><td></td></tr><tr><td>Micropipette</td><td>Origio, Denmark</td><td>MBB-FP-L-0</td><td></td></tr><tr><td>Syringe pumps</td><td>KD Scientific</td><td>KDS-210</td><td></td></tr><tr><td>100 &amp;mu;L syringe</td><td>Hamilton</td><td>81020</td><td>Gas tight, PTFE Luer lock</td></tr><tr><td>1 mL syringe</td><td>Hamilton</td><td>81327</td><td>Gas tight, PTFE Luer lock</td></tr><tr><td>Silicone isolator</td><td>Grace Bio-Labs</td><td>JTR24R-A-0.5</td><td>6x4 well silicone isolator with adhesive</td></tr><tr><td>Laser cutter</td><td>VersaLASE</td><td>VLS2.30 CO2 Laser 3W</td><td>for laser cutting of custom isolators</td></tr><tr><td>1mm thick acrylic sheet</td><td>Weatherall-UK</td><td>Clarex Precision Sheet 001</td><td>for laser cutting of custom isolators</td></tr><tr><td>Adhesive sheet</td><td>3M</td><td></td><td>used to adhere custom isolators to microarrayed coverslips</td></tr><tr><td>Super glue</td><td>Loctite</td><td>LOCPFG3T</td><td></td></tr><tr><td>150 &amp;mu;m ID/360 &amp;mu;m OD fused silica tubing</td><td>IDEX</td><td>FS-115</td><td></td></tr><tr><td>1.0 mm ID/1/16&amp;rdquo; OD PFA tubing</td><td>IDEX</td><td>1503</td><td></td></tr><tr><td>0.014&amp;rdquo; ID/0.062&amp;rdquo; OD PTFE tubing</td><td>Kinesis</td><td>008T16-100</td><td></td></tr><tr><td>1.0 mm ID/2.0 mm OD FEP tubing</td><td>IDEX</td><td>1673</td><td></td></tr><tr><td>Bovine Serum Albumen (BSA)</td><td>Fisher Scientific</td><td>BP9700100</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M5904</td><td></td></tr><tr><td>Ultra-pure water</td><td>Millipore, Germany</td><td>MilliQ</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopy &amp;amp; Optics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TIRF microscope with encoded XY stage</td><td>Nikon, Japan</td><td>Nikon Ti-E</td><td></td></tr><tr><td>EM-CCD</td><td>Andor Technologies, Ireland</td><td>IXON DU-897E</td><td></td></tr><tr><td>Laser excitation source</td><td>Vortran, USA</td><td>Stradus 488-50</td><td></td></tr><tr><td>Optical lysis laser source</td><td>Continuum, USA</td><td>Surelite SLI-10</td><td></td></tr><tr><td>Microscope filter cube for TIRF</td><td>Chroma, USA</td><td>z488bp</td><td></td></tr><tr><td>Microscope filter cube for Optical Lysis</td><td>Laser 2000, UK</td><td>LPD01-532R-25</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fiji</td><td>Open Source</td><td></td><td>Image analysis software</td></tr><tr><td>Matlab</td><td>Mathworks</td><td>version 7.14 or higher</td><td>Image analysis software</td></tr></tbody></table>

counting proteins in single cells with addressable droplet microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

The absolute basal protein copy number of p53 was determined with single cell resolution in a human colon cancer cell line, BE cells. We demonstrate how p53 expression can vary over several orders of magnitude and show a weakly positive correlation between cell size and protein copy number within the resting BE cell population.
Addressable Droplet Microarrays are formed when aqueous droplets are dispensed at antibody spot locati...

Watch this Scientific Journal Video about Counting Proteins in Single Cells with Addressable Droplet Microarrays at JoVE.com

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

counting-proteins-in-single-cells-with-addressable-droplet-microarrays

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

8.5K Views.  Imperial College London. Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Video: Counting Proteins in Single Cells with Addressable Droplet Microarrays

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengineering

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

Chemistry

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Biochemistry

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Microfluidic Droplet Assay for Single-Cell Cytokine Secretion Analysis

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

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.

Author Spotlight: Unveiling the Polyfunctionality and Heterogeneity in Immune Responses

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. ...

Immunology and Infection

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Biology

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.