Eprints.whiterose.ac.uk



Quantifying single cell secretion in real time using resonant hyperspectral imaging José Juan-Colás1,2*, Ian S. Hitchcock3, Mark Coles4, Steven Johnson2 and Thomas F. Krauss11 Department of Physics, University of York, Heslington, York YO10 5DD, UK2 Department of Electronic Engineering, University of York, Heslington, York YO10 5DD, UK3 Department of Biology, University of York, Heslington, York YO10 5DD, UK4 Kennedy Institute of Rheumatology, University of Oxford, Headington, Oxford OX3 7FY, UK*Correspondence: (J.J-C) jose.juancolas@york.ac.uk ORCID ID:?0000-0002-1031-915XAbstractCell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro and real-time analysis of specific single-cell signalling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet de-sialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.Keywords: photonic biosensing, single-cell analysis, label-free SignificanceCell communication is primarily regulated by secreted proteins. However, inhomogeneous secretion may indicate commencement of disease. Therefore, the ability to parallelly monitor innate individual cell secretion kinetics is crucial to understand systemic malfunctions. Here, we report a high-throughput method for parallel, in vitro and real-time analysis of specific single-cell signalling using hyperspectral photonic crystal resonant technology without the need of adding any fluorescent label. We mapped the secretion of a signalling protein (TPO) from individual human HepG2 cells and for the first time quantify the heterogeneity in TPO expression as a function of the desialylated (aged) platelet level concentration. Given its ease of use and implementation, we anticipate that our technology will transform single-cell protein expression analysis for applications beyond basic research.\bodyProteins that are secreted from cells into the extracellular matrix make up 13-20% of the entire human proteomeADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/J.BBAPAP.2013.01.022", "ISSN" : "1570-9639", "abstract" : "The cell secretome is a collection of proteins consisting of transmembrane proteins (TM) and proteins secreted by cells into the extracellular space. A significant portion (~13\u201320%) of the human proteome consists of secretory proteins. The secretory proteins play important roles in cell migration, cell signaling and communication. There is a plethora of methodologies available like Serial Analysis of Gene Expression (SAGE), DNA microarrays, antibody arrays and bead-based arrays, mass spectrometry, RNA sequencing and yeast, bacterial and mammalian secretion traps to identify the cell secretomes. There are many advantages and disadvantages in using any of the above methods. This review aims to discuss the methodologies available along with their potential advantages and disadvantages to identify secretory proteins. This review is a part of a Special issue on The Secretome. This article is part of a Special Issue entitled: An Updated Secretome.", "author" : [ { "dropping-particle" : "", "family" : "Mukherjee", "given" : "Paromita", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mani", "given" : "Sridhar", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics", "id" : "ITEM-1", "issue" : "11", "issued" : { "date-parts" : [ [ "2013", "11", "1" ] ] }, "page" : "2226-2232", "publisher" : "Elsevier", "title" : "Methodologies to decipher the cell secretome", "type" : "article-journal", "volume" : "1834" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(1)", "plainTextFormattedCitation" : "(1)", "previouslyFormattedCitation" : "¹" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(1). These secreted proteins typically function as mediators of cell-cell signalling and cellular proliferation and thus play an important role in a wide variety of important physiological and pathological processes. A prime example of this is haematopoiesis, which is tightly regulated by the paracrine or endocrine secretion of cytokines and hormones to maintain haematopoietic stem cells and drive specific lineage differentiation. Impairment of this regulatory system, through organ failure or deleterious mutations, can result in malignant overproduction of blood cells, aberrant immune function or bone marrow failure. Current understanding of secreted proteins is based largely on traditional proteomic analysis, such as enzyme-linked immunosorbent assays (ELISA) and mass spectrometry (MS), of proteins in the extracellular medium. While these techniques provide the high sensitivity required to detect low concentrations of proteins secreted into the surrounding matrix, a comprehensive understanding of protein secretion also requires tools with sufficient resolution to map the temporal and spatial regulation of secretion. For example, cytokine secretion by innate immune cells is orchestrated between cells to provide an initial inflammatory or allergic response and later to ensure this response subsides in a timely and coordinated fashion. Traditional ELISA and MS both lack the spatial and temporal resolution required to map the kinetics of such tightly regulated and orchestrated protein secretion. Furthermore, single cell analytical techniques have shown that individual cells with apparently identical phenotype, exhibit heterogeneity in protein secretion. Thus, the ability to interrogate and monitor biological systems at the single-cell level provides a precise route for determining which variation is random and which is meaningful, and for building better models of the behavior of individual cells. Again, ELISA and MS typically measure the average response of a cell-population at a fixed time point and are thus unable to screen the “hidden world” beneath population averagesADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.cell.2010.04.033", "ISBN" : "1097-4172 (Electronic)\\r0092-8674 (Linking)", "ISSN" : "00928674", "PMID" : "20478246", "abstract" : "A central challenge of biology is to understand how individual cells process information and respond to perturbations. Much of our knowledge is based on ensemble measurements. However, cell-to-cell differences are always present to some degree in any cell population, and the ensemble behaviors of a population may not represent the behaviors of any individual cell. Here, we discuss examples of when heterogeneity cannot be ignored and describe practical strategies for analyzing and interpreting cellular heterogeneity. \u00a9 2010 Elsevier Inc.", "author" : [ { "dropping-particle" : "", "family" : "Altschuler", "given" : "Steven J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wu", "given" : "Lani F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "559-563", "title" : "Cellular Heterogeneity: Do Differences Make a Difference?", "type" : "article-journal", "volume" : "141" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(2)", "plainTextFormattedCitation" : "(2)", "previouslyFormattedCitation" : "²" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(2). Robust and non-disruptive methodologies to profile protein secretion at the single cell level and to map changes in secretion due to cooperative interactions or exposure to therapeutics are critical if we are to fully understand their role in both healthy and diseased states.Spatial confinement provided by microfluidic systems have been exploited for molecular analysis and the single cell level and sub-nanolitre microwell arrays have recently been employed to analyse protein secretion at single-cell resolution.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/srep04736", "ISSN" : "20452322", "PMID" : "24751898", "abstract" : "Protein secretion, a key intercellular event for transducing cellular signals, is thought to be strictly regulated. However, secretion dynamics at the single-cell level have not yet been clarified because intercellular heterogeneity results in an averaging response from the bulk cell population. To address this issue, we developed a novel assay platform for real-time imaging of protein secretion at single-cell resolution by a sandwich immunoassay monitored by total internal reflection microscopy in sub-nanolitre-sized microwell arrays. Real-time secretion imaging on the platform at 1-min time intervals allowed successful detection of the heterogeneous onset time of nonclassical IL-1\u03b2 secretion from monocytes after external stimulation. The platform also helped in elucidating the chronological relationship between loss of membrane integrity and IL-1\u03b2 secretion. The study results indicate that this unique monitoring platform will serve as a new and powerful tool for analysing protein secretion dynamics with simultaneous monitoring of intracellular events by live-cell imaging.", "author" : [ { "dropping-particle" : "", "family" : "Shirasaki", "given" : "Yoshitaka", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamagishi", "given" : "Mai", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Nobutake", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Izawa", "given" : "Kazushi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nakahara", "given" : "Asahi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mizuno", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shoji", "given" : "Shuichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Heike", "given" : "Toshio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Harada", "given" : "Yoshie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nishikomori", "given" : "Ryuta", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ohara", "given" : "Osamu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Scientific Reports", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2014" ] ] }, "title" : "Real-time single-cell imaging of protein secretion", "type" : "article-journal", "volume" : "4" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(3)", "plainTextFormattedCitation" : "(3)", "previouslyFormattedCitation" : "³" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(3) Here, protein detection was achieved using a sandwich immunoassay with a fluorescently labelled detection antibody in which the fluorophore was excited by total internal reflection microscopy. While providing insight into the kinetics of protein secretion from single cells, the binding of the detection antibody to form sandwich immunocomplexes is the rate limiting step, ultimately limiting the temporal resolution of this approach. Furthermore, the need to spatially confine single cells within individual microwells precludes the possibility of mapping protein secretion in cellular communities in which protein secretion is orchestrated between interacting cells. More recently, an optofluidic approach based on nanoplasmonic sensing has been proven as a specific and sensitive technique to measure cytokine secretion at the single-cell levelADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1002/smll.201800698", "ISSN" : "16136829", "abstract" : "\u00a9 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Single-cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single-cell resolution reveals the real-time functional status of individual cells. Fluorescent and colorimetric-based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label-free optofluidic nanoplasmonic biosensor is introduced for single-cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin-2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single-cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single-cell signaling for basic and clinical research.", "author" : [ { "dropping-particle" : "", "family" : "Li", "given" : "Xiaokang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Soler", "given" : "Maria", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Szydzik", "given" : "Crispin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Khoshmanesh", "given" : "Khashayar", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schmidt", "given" : "Julien", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Coukos", "given" : "George", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mitchell", "given" : "Arnan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Altug", "given" : "Hatice", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Small", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2018" ] ] }, "page" : "1-11", "title" : "Label-Free Optofluidic Nanobiosensor Enables Real-Time Analysis of Single-Cell Cytokine Secretion", "type" : "article-journal", "volume" : "1800698" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(4)", "plainTextFormattedCitation" : "(4)", "previouslyFormattedCitation" : "⁴" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(4). While overcoming the need for a detection antibody due to its intrinsic ultra-sensitivity (detection limit in the pg/mL range), this spectroscopic approach is designed to report protein secretion from an isolated single-cell, thus is not able to map orchestrated protein secretion in cellular communities or provide high throughput analysis of secretion dynamics. Similarly, McDonald et al. demonstrated an alternative approach for the real-time detection of secreted proteins based on interferometric detection of scattered light (iSCAT)ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.nanolett.7b04494", "ISSN" : "15306992", "PMID" : "29227108", "abstract" : "Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT), and is demonstrated with Laz388 cells\u2014an Epstein-Barr virus (EBV) transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa \u2013 1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general, and as we show, can also be applied to studying the lysate of a single cell. Our results establish label...", "author" : [ { "dropping-particle" : "", "family" : "McDonald", "given" : "Matthew P.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gemeinhardt", "given" : "Andr\u00e9", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "K\u00f6nig", "given" : "Katharina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliarik", "given" : "Marek", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schaffer", "given" : "Stefanie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "V\u00f6lkl", "given" : "Simon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Aigner", "given" : "Michael", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mackensen", "given" : "Andreas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sandoghdar", "given" : "Vahid", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nano Letters", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2018" ] ] }, "page" : "513-519", "title" : "Visualizing Single-Cell Secretion Dynamics with Single-Protein Sensitivity", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(5)", "plainTextFormattedCitation" : "(5)", "previouslyFormattedCitation" : "⁵" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(5). While this removed the limitations imposed by fluorescently labelled antibodies and microfluidic confinement, the lack of immunoreagents limits the specificity of the approach; iSCAT is sensitive to individual proteins with molecular weights in the range 100?1100 kDa, but lacks specificity to differentiate between proteins within this range. Furthermore, the field of view was here limited to only 6 ?m, again unable to report parallel secretion from single cells within large populations. Here, we present a simple, yet powerful technology to monitor and quantify orchestrated cell-signaling at the single cell level, in real-time and in a label-free manner using photonic crystal resonant imaging. Photonic crystal structures, including 1D gratings, have recently gained considerable attention as they offer the same evanescence wave sensing principleADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1088/2040-8986/aac75b", "ISSN" : "2040-8978", "author" : [ { "dropping-particle" : "", "family" : "Pitruzzello", "given" : "Giampaolo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Krauss", "given" : "Thomas F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Optics", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2018", "5", "23" ] ] }, "title" : "Photonic crystal resonances for sensing and imaging", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(6)", "plainTextFormattedCitation" : "(6)", "previouslyFormattedCitation" : "⁶" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(6) and performance of surface plasmon resonant (SPR) sensors without the need of complex instrumentation e.g. bulk prism-coupling instrumentation, allowing further integration with diverse laboratory based apparatus, such as phase contrast and fluorescent microscopy. Photonic crystal resonant surfaces (PCRS) consisting of silicon nitride (Si3N4), or titanium dioxide (TiO2), have been demonstrated that allow label-free cellular imaging with subcellular spatial resolution (resolution 2-6 ?m depending on the device orientation) through resonant wavelength based hyperspectral imagingADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1109/JPHOT.2015.2435699", "ISSN" : "19430655", "PMID" : "26356353", "abstract" : "By depositing a resolution test pattern on top of a Si3N4 photonic crystal resonant surface, we have measured the dependence of spatial resolution on refractive index contrast \u0394n. Our experimental results and finite-difference time-domain (FDTD) simulations at different refractive index contrasts show that the spatial resolution of our device reduces with reduced contrast, which is an important consideration in biosensing, where the contrast may be of order 10-2. We also compare 1-D and 2-D gratings, taking into account different incidence polarizations, leading to a better understanding of the excitation and propagation of the resonant modes in these structures, as well as how this contributes to the spatial resolution. At \u0394n 0.077, we observe resolutions of 2 and 6 \u03bcm parallel to and perpendicular to the grooves of a 1-D grating, respectively, and show that for polarized illumination of a 2-D grating, resolution remains asymmetrical. Illumination of a 2-D grating at 450 results in symmetric resolution. At very low index contrast, the resolution worsens dramatically, particularly for \u0394n <; 0.01, where we observe a resolution exceeding 10 \u03bcm for our device. In addition, we measure a reduction in the resonance linewidth as the index contrast becomes lower, corresponding to a longer resonant mode propagation length in the structure and contributing to the change in spatial resolution.", "author" : [ { "dropping-particle" : "", "family" : "Triggs", "given" : "G. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fischer", "given" : "M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stellinga", "given" : "D.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scullion", "given" : "M. G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Evans", "given" : "G. J O", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Krauss", "given" : "T. F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "IEEE Photonics Journal", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2015" ] ] }, "title" : "Spatial resolution and refractive index contrast of resonant photonic crystal surfaces for biosensing", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(7)", "plainTextFormattedCitation" : "(7)", "previouslyFormattedCitation" : "⁷" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(7) (see Methods). Essentially, PCRS exhibit a spatially confined resonant wave at specific wavelengths, which interferes constructively or destructively with the incoming light beam, leading to a minima (maxima) in the transmission (reflection) spectrum (see Supplementary Information). Moreover, PCRS can be fabricated over large areas to provide a high field-of-view (the order of centimeters), to allow parallel imaging of multiple cells without affecting the intrinsic spatial resolution. As the evanescent field is confined close to the grating surface (penetration depth ~200 nm), the sensors are highly sensitive not just to large refractive index changes as a consequence of cell attachment to the sensor surface (Figure 1a), but also to smaller, local refractive index changes such as those originating from protein-ligand binding events occurring on the sensor surface, all in real-time, label-free and a quantifiable mannerADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1002/lpor.201100025", "ISBN" : "1094-4087", "ISSN" : "18638880", "abstract" : "The application of portable, easy-to-use and highly sensitive lab-on-a-chip biosensing devices for real-time diag- nosis could offer significant advantages over current analytical methods. Integrated optics-based biosensors have become the most suitable technology for lab-on-chip integration due to their ability for miniaturization, their extreme sensitivity, robustness, reliability, and their potential for multiplexing and mass produc- tion at low cost. This review provides an extended overview of the state-of-the-art in integrated photonic biosensors technology including interferometers, grating couplers, microring resonators, photonic crystals and other novel nanophotonic transducers. Particular emphasis has been placed on describing their real biosensing applications and wherever possible a comparison of the sensing performances between each type of device is included. The way towards achieving operative lab-on-a-chip platform incorporating the photonic biosensors is also reviewed. Concluding remarks regarding the future prospects and poten- tial impact of this technology are also provided.", "author" : [ { "dropping-particle" : "", "family" : "Estevez", "given" : "M. C.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Alvarez", "given" : "M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lechuga", "given" : "Laura M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Laser & Photonics Reviews", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "463-487", "title" : "Integrated optical devices for lab-on-a-chip biosensing applications", "type" : "article-journal", "volume" : "6" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(8)", "plainTextFormattedCitation" : "(8)", "previouslyFormattedCitation" : "⁸" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(8). By functionalizing the photonic crystal sensor surface with capture molecules (e.g. antibodies), the surface can thus be rendered sensitive to specific biomolecules secreted by the cells, enabling mapping of protein secretion gradients at the single-cell level without the need of a fluorescent label. Instead of immobilizing antibodies such that they are ordered on a single plane on the sensor surface, we increased the density of antibodies within the evanescence wave of the PCRS, and thus the detection sensitivity, by creating a 3D network using branched glucan dextran (see Methods). The surface was finally treated with purified casein to inhibit non-specific binding of other molecules secreted into the extracellular matrix (see Methods and Supplementary Information) (Figure 1b). This blocking approach allowed us to maximise the signal-to-noise ratio of our detection system and perform long-term analysis of cellular activities under appropriate conditions.To demonstrate the performance of our technology, we first mapped the secretion of thrombopoietin (TPO) from an immortal cell line comprised of baby hamster kidney cells (BHK). TPO is a haematopoietic cytokine essential for driving the differentiation of megakaryocytes and maintaining physiological levels of circulating platelets. Additionally, TPO is one of only a small number of cytokines responsible for maintaining haematopoietic stem cells. The TPO secreting BHK cells (BHK-TPOADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1074/jbc.M201297200", "abstract" : "Thrombopoietin (TPO), the primary regulator of platelet production, is composed of an amino-terminal 152 amino acids, sufficient for activity, and a carboxyl-terminal region rich in carbohydrates (183 residues) that enhances secretion of the molecule. Full-length TPO is secreted at levels 10\u201320-fold greater than truncated TPO. By introducing into mammalian cells a novel cDNA encoding the TPO secretory leader linked to its carboxyl-terminal domain (TPO glycan domain (TGD)), we tested whether TGD could function in trans to enhance secretion of TPO. The artificial TGD was secreted, inactive in proliferation assays, and did not inhibit TPO activity. However, when co-transfected with a cDNA encoding truncated TPO, TGD enhanced secretion 4-fold, measured by specific bioassay and immunoassay. TGD also enhanced secretion of granulocyte monocyte colony-stimulating factor and stem cell factor but did not affect the production of erythropoietin, interleukin-3, growth hormone, or of full-length TPO. To localize TGD function, we added an endoplasmic reticulum (ER) retention signal to TGD and, separately, deleted the secretory leader. Deletion of the secretory leader attenuated the secretory function of TGD, whereas addition of the ER retention signal did not alter its function. To investigate the physiologic role of TGD in folding and proteasomal protection, we tested full-length and truncated TPO in assays of protein refolding, and we examined protein stability in the presence of proteasome inhibitors. We found that truncated TGD re-folds readily and that proteasome-mediated degradation contributes to the poor secretion of truncated TPO. We conclude that TGD enhances secretion of TPO and can additionally function as an inter-molecular chaperone, in part because of its ability to prevent degradation of the hormone. The cellular location of TGD action is likely to be within the ER or earlier in the secretory pathway. ", "author" : [ { "dropping-particle" : "", "family" : "Linden", "given" : "Hannah M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kaushansky", "given" : "Kenneth", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Biological Chemistry ", "id" : "ITEM-1", "issue" : "38 ", "issued" : { "date-parts" : [ [ "2002", "9", "20" ] ] }, "note" : "10.1074/jbc.M201297200", "page" : "35240-35247", "title" : "The Glycan Domain of Thrombopoietin (TPO) Acts intrans to Enhance Secretion of the Hormone and Other Cytokines ", "type" : "article-journal", "volume" : "277 " }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(9)", "plainTextFormattedCitation" : "(9)", "previouslyFormattedCitation" : "⁹" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(9)) used here constitutively release TPO without the need for an extracellular triggering biomolecule (see Methods and Supplementary Information). The BHK-TPO cells are also adherent, and assemble in a monolayer on the sensor surface, which here was functionalized with antibodies selective against TPO (see Methods) to map the secretion of TPO from individual BHK-TPO cells. A resonant image of the PCRS prior to cell adhesion (i.e. PCRS functionalized with dextran, anti-TPO antibodies and casein) was recorded (Figure 1d) over a 55 x 40 ?m region of interest and the fraction of pixels resonant at a specific wavelength plotted on a histogram (Figure 1c). The mean resonance wavelength of the functionalized PCRS was subsequently fixed to 0 nm to provide a baseline resonant image. The width of this Gaussian distribution (± 0.5 nm) is largely a consequence of inhomogeneity in the surface chemistry plus experimental error and thus a threshold of 1 nm was set. Shifts in the resonance wavelength above this threshold arise due to local changes in refractive index due to binding of BHK-TPO cells/ TPO to the surface. The functionalized PCRS was subsequently exposed to BHK-TPO cells suspended in the supporting culture media (concentration of 105 U/mL; significantly below the 100% confluence level to avoid contact inhibition of inherent protein/gene expression) and the adhesion of a single BHK-TPO cell in our defined region of interest was recorded using hyperspectral imaging. The local increase in refractive index associated with the adhesion of a single cell to the PCRS induced a change in the resonance wavelength over the region of interest (Figure 1g) which again can be quantified by the histogram of the fraction of pixels resonant at a specific wavelength (Figure 1f). Subsequently, resonant images of the region of interest collected at 3-minute time intervals showed a shift in the resonance frequency around the periphery of the immobilized cell, corresponding to an increase in the local refractive index, the spatial extent of which increased with time. This can be seen in the contour plot of Figure 1i (also see Supplementary Information), which shows the spatial position over the region of interest at which the resonance wavelength increases beyond the 1 nm threshold. This increase in resonant frequency around the cell is associated with the local increase in refractive index due to TPO secreted from the BHK cell binding to the anti-TPO antibodies immobilized on the PCRS. This is further confirmed by the absence of a shift in resonant frequency in control experiments where the surface was functionalized with an antibody which lacks specificity to TPO (here, the surface was functionalised with antibodies specific for immunoglobulin G, IgG) or when using BHK-TPO cells in which the TPO secretion has been supressed using the protein transport inhibitor brefeldin A (Figure 2f and g). To aid comparison between experiments and quantify the kinetics of protein secretion from a single cell, we define the total secretion area covered by the cell and surface bound TPO (i.e. the area equivalent to a shift in resonant frequency 1 nm above the background resonance). To further calibrate the measurement, we subsequently subtract the region relative to cell attachment from this total secretion area, which is statistically delimited by a shift in resonant frequency of 1.7 nm ± 0.1 nm (see Supplementary Information). Figure 1j shows the change in total secretion area covered by the contour plot as a function of time. Assuming the secreted TPO bind to the surface immobilized antibody as a single adsorption process, the time evolution of this secretion process can be modelled as a Langmuir adsorption distribution yielding a single cell secretion rate of 22 ?m2/h (coefficient of determination R2 of 0.94 ± 0.04) (see Methods). The ability to image and map protein secretion from multiple single cells is critical to understanding heterogeneity of cell populations. The wide field of view provided by our technology enables multiple, parallel measurements of single cells in real-time to provide insight into such complex cellular processes. We extended the region of interest to an area of 500 x 500 ?m (0.25 mm2) to characterise the dynamics and the kinetics of a population of 30 individual BHK-TPO cells without compromising our sub-cellular spatial resolutionADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1109/JPHOT.2015.2435699", "ISSN" : "19430655", "PMID" : "26356353", "abstract" : "By depositing a resolution test pattern on top of a Si3N4 photonic crystal resonant surface, we have measured the dependence of spatial resolution on refractive index contrast \u0394n. Our experimental results and finite-difference time-domain (FDTD) simulations at different refractive index contrasts show that the spatial resolution of our device reduces with reduced contrast, which is an important consideration in biosensing, where the contrast may be of order 10-2. We also compare 1-D and 2-D gratings, taking into account different incidence polarizations, leading to a better understanding of the excitation and propagation of the resonant modes in these structures, as well as how this contributes to the spatial resolution. At \u0394n 0.077, we observe resolutions of 2 and 6 \u03bcm parallel to and perpendicular to the grooves of a 1-D grating, respectively, and show that for polarized illumination of a 2-D grating, resolution remains asymmetrical. Illumination of a 2-D grating at 450 results in symmetric resolution. At very low index contrast, the resolution worsens dramatically, particularly for \u0394n <; 0.01, where we observe a resolution exceeding 10 \u03bcm for our device. In addition, we measure a reduction in the resonance linewidth as the index contrast becomes lower, corresponding to a longer resonant mode propagation length in the structure and contributing to the change in spatial resolution.", "author" : [ { "dropping-particle" : "", "family" : "Triggs", "given" : "G. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fischer", "given" : "M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stellinga", "given" : "D.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scullion", "given" : "M. G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Evans", "given" : "G. J.O.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Krauss", "given" : "T. F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "IEEE Photonics Journal", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2015" ] ] }, "title" : "Spatial resolution and refractive index contrast of resonant photonic crystal surfaces for biosensing", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(10)", "plainTextFormattedCitation" : "(10)", "previouslyFormattedCitation" : "¹⁰" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(10). We employed the same protocol described previously for a single BHK-TPO cell to quantify protein secretion from multiple cells within the new region of interest. A threshold of 1nm was again chosen to define the background resonance and the increase in resonance around each cell was monitored every 3 minutes for a duration of 130 min (Figure 2e N.B. for clarity a region of interest of 160 x 210 ?m is shown at timepoints 0, 8, 15 and 23 min). To demonstrate the specificity of our assay we also challenged our PCRS sensor with two different control systems. In the first control, the PCRS was functionalised with a control antibody and challenged with BHK-TPO cells (Figure 2f). The second control comprised a surface functionalised with anti-TPO challenged with a population of BHK-TPO cells treated with the protein transport inhibitor brefeldin A to supress secretion of TPO (Figure 2g) (see Methods). Secretion of TPO and subsequent binding to surface immobilized anti-TPO led to an increase in the resonant frequency around each of 30 BHK-TPO cells (Figure 2a). After 120 min, TPO secreted from the BHK-TPO cells had diffused isotropically away from each cell over an average secretion area of ?anti-TPO ~ 720 ?m2 around each cell. In contrast, the average shift in resonant frequency around individual cells in the control experiments was minimal due to the inhibition of TPO secretion from BHK-TPO cells treated with brefeldin A and the lack of specificity of the anti-IgG sensor for TPO (Figure 2h). The wide field of view coupled with high spatial resolution allows statistical analysis of protein secretion from individual cells. Histograms of surface adsorption for each BHK-TPO cell (Figure 2i) revealed a high degree of heterogeneity in terms of TPO secretion (standard deviation of ?anti-TPO ~ 120 ?m2). This contrasts the control system treated with brefeldin A which exhibits a lower standard deviation (?brefeldin A ~ 60 ?m2) due to the homogeneity of the non-secreting system. The heterogeneity of protein secretion was also observed in the kinetics of TPO secretion (See Methods, Figure 2i). The average rate of protein secretion from BHK-TPO cells and adsorption onto surface immobilized anti-TPO was κanti-TPO ~ 22 ?m2/h (coefficient of determination R2 of 0.94 ± 0.04), significantly higher than the kinetics of non-specific adsorption observed in the control systems (~ 9 ?m2/h and ~ 6.5 ?m2/h for κanti-IgG and κbrefeldin A, respectively). The real-time and label-free capabilities associated with our analytical technology not only enables high throughput analysis at the single cell level but also the ability to study single cell-dynamics in clinical samples, for example the personalized response of patients’ cells to drugs or other treatments. To highlight this capability, we have used this this technology to determine the effects of human platelets on mediating TPO release at the single cell level. Maintaining physiological levels of circulating platelets relies on a finely tuned balance between platelet generation and clearance. Although TPO is the key regulator of platelet production, the specific mechanisms that underpin TPO production and this physiological feedback loop remain unclearADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "abstract" : "Thrombopoietin (c-Mpl ligand) has recently been purified and is considered to be the humoral regulator of platelet production. To see whether this molecule possessed the physiologic characteristics necessary to mediate the feed-back loop between blood platelets and the bone marrow megakaryocytes, we determined the relationship between blood levels of thrombopoietin and changes in the circulating platelet mass. We developed a model of nonimmune thrombocytopenia in rabbits by the subcutaneous administration of busulfan. Compared with pretreatment plasma, plasma taken from all thrombocytopenic rabbits at their platelet nadir contained increased amounts of thrombopoietin. All of this activity was neutralized by soluble c-Mpl receptor. We subsequently measured the level of thrombopoietin in the circulation over the entire time course after the administration of busulfan. As the platelet mass declined, levels of thrombopoietin increased inversely and proportionally and peaked during the platelet nadir. With return of the platelet mass toward normal, thrombopoietin levels decreased accordingly. When platelets were transfused into thrombocytopenic rabbits near the time of their platelet count nadir, the elevated levels of thrombopoietin decreased. In addition, platelets were observed to remove thrombopoietin from thrombocytopenic plasma in vitro. These results confirm that thrombopoietin is the humoral mediator of megakaryocytopoiesis and suggest that the platelet mass may directly play a role in regulating the circulating levels of this factor.", "author" : [ { "dropping-particle" : "", "family" : "Kuter", "given" : "D J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rosenberg", "given" : "R D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Blood", "id" : "ITEM-1", "issue" : "10", "issued" : { "date-parts" : [ [ "1995", "5", "15" ] ] }, "page" : "2720 LP - 2730", "title" : "The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit", "type" : "article-journal", "volume" : "85" }, "uris" : [ "", "" ] }, { "id" : "ITEM-2", "itemData" : { "abstract" : "The involvement of platelets and the c-mpl receptor in the regulation of thrombopoietin (TPO) plasma concentrations and tissue mRNA levels was investigated in both normal mice and mice defective in c-mpl (c-mpl- /-). Although c-mpl-/- mice have fewer platelets and higher plasma TPO activity than normal mice, there was no increase in TPO mRNA levels as measured by an S1 nuclease protection assay. After the intravenous injection of 125I-TPO, specific uptake of radioactivity by the spleen and blood cells was present in the normal mice, but absent in the c-mpl- /- mice. Platelet-rich plasma (PRP) from normal mice was able to bind and internalize 125I-TPO, whereas PRP from c-mpl-/- mice lacked this ability. Analysis of 125I-TPO binding to normal PRP indicated that binding was specific and saturable, with an approximate affinity of 560 pmol/L and 220 receptors per platelet. PRP from normal mice was also able to degrade 125I-TPO into lower molecular weight fragments. After the intravenous injections, c-mpl-/- mice cleared a dose of 125I-TPO at a much slower rate than did normal mice. Injection of washed platelets from normal mice into c-mpl-/- mice resulted in a dramatic, but transient, decrease in plasma TPO levels. These data provide evidence that platelets regulate plasma TPO levels via binding to the c-mpl receptor on circulating platelets.", "author" : [ { "dropping-particle" : "", "family" : "Fielder", "given" : "P J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gurney", "given" : "A L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stefanich", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Marian", "given" : "M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Moore", "given" : "M W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Carver-Moore", "given" : "K", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sauvage", "given" : "F J", "non-dropping-particle" : "de", "parse-names" : false, "suffix" : "" } ], "container-title" : "Blood", "id" : "ITEM-2", "issue" : "6", "issued" : { "date-parts" : [ [ "1996", "3", "15" ] ] }, "page" : "2154 LP - 2161", "title" : "Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets", "type" : "article-journal", "volume" : "87" }, "uris" : [ "", "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1046/j.1365-2141.1999.01459.x", "ISSN" : "0007-1048", "abstract" : "Little is known about the behaviour of endogenous thrombopoietin (TPO) serum levels during rapid sequences of dose?intensified chemotherapy. To characterize the relationship between TPO levels and platelet counts in this setting we serially measured both parameters over the entire treatment period of patients receiving multicycle polychemotherapy. We found TPO and platelet responses to be generally antagonistic through all cycles. However, a cross?correlation analysis indicated that TPO responses preceded platelet responses by approximately one day in all patients. The cumulative severity of thrombocytopenia observed over successive cycles was accompanied by an increasing TPO response which tended to grow overproportionally in relation to the degree of peripheral thrombocytopenia. These findings are consistent with a model suggesting that both platelet and megakaryocyte mass contribute to a receptor?dependent consumption process regulating the endogenous TPO level. In order to develop optimal schedules for exogenous TPO administration it might be important to consider endogenous TPO response characteristics.", "author" : [ { "dropping-particle" : "", "family" : "Christoph", "given" : "Engel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Markus", "given" : "Loeffler", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Horst", "given" : "Franke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stephan", "given" : "Schmitz", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "British Journal of Haematology", "id" : "ITEM-3", "issue" : "3", "issued" : { "date-parts" : [ [ "2001", "12", "25" ] ] }, "note" : "doi: 10.1046/j.1365-2141.1999.01459.x", "page" : "832-838", "publisher" : "Wiley/Blackwell (10.1111)", "title" : "Endogenous thrombopoietin serum levels during multicycle chemotherapy", "type" : "article-journal", "volume" : "105" }, "uris" : [ "", "" ] } ], "mendeley" : { "formattedCitation" : "(11\u201313)", "plainTextFormattedCitation" : "(11\u201313)", "previouslyFormattedCitation" : "^11\u201313" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(11–13). Recent experiments suggest that TPO levels are directly dependent on the concentration of circulating, desialylated (aged) plateletsADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nm.3770", "ISBN" : "1546-170X (Electronic)\\r1078-8956 (Linking)", "ISSN" : "1546170X", "PMID" : "25485912", "abstract" : "The hepatic Ashwell-Morell receptor (AMR) can bind and remove desialylated platelets. Here we demonstrate that platelets become desialylated as they circulate and age in blood. Binding of desialylated platelets to the AMR induces hepatic expression of thrombopoietin (TPO) mRNA and protein, thereby regulating platelet production. Endocytic AMR controls TPO expression through Janus kinase 2 (JAK2) and the acute phase response signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. Recognition of this newly identified physiological feedback mechanism illuminates the pathophysiology of platelet diseases, such as essential thrombocythemia and immune thrombocytopenia, and contributes to an understanding of the mechanisms of thrombocytopenia observed with JAK1/2 inhibition.", "author" : [ { "dropping-particle" : "", "family" : "Grozovsky", "given" : "Renata", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Begonja", "given" : "Antonija Jurak", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Liu", "given" : "Kaifeng", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Visner", "given" : "Gary", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hartwig", "given" : "John H.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Falet", "given" : "Herv\u00e9", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hoffmeister", "given" : "Karin M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature Medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "47-54", "title" : "The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling", "type" : "article-journal", "volume" : "21" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(14)", "plainTextFormattedCitation" : "(14)", "previouslyFormattedCitation" : "¹⁴" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(14). However, the levels of TPO protein upregulation and the kinetics of TPO secretion at the single cell level have yet to be quantified. We have thus applied our method to answer these important questions. We studied platelet-mediated regulation and secretion of TPO from exemplar human cells (HepG2 cell line human hepatocyte carcinoma, see Methods) over a 500 x 500 ?m2 region of interest. TPO secretion was monitored using a PCRS functionalized with anti-TPO and recorded as a function of exposure to human platelets with differing levels of desialylationADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1134/S0006297913070067", "ISSN" : "1608-3040", "abstract" : "Terminal sialic acid residues are found in abundance in glycan chains of glycoproteins and glycolipids on the surface of all live cells forming an outer layer of the cell originally known as glycocalyx. Their presence affects the molecular properties and structure of glycoconjugates, modifying their function and interactions with other molecules. Consequently, the sialylation state of glycoproteins and glycolipids has been recognized as a critical factor modulating molecular recognitions inside the cell, between the cells, between the cells and the extracellular matrix, and between the cells and certain exogenous pathogens. Until recently sialyltransferases that catalyze transfer of sialic acid residues to the glycan chains in the process of their biosynthesis were thought to be mainly responsible for the creation and maintenance of a temporal and spatial diversity of sialylated moieties. However, the growing evidence suggests that in mammalian cells, at least equally important roles belong to sialidases/neuraminidases, which are located on the cell surface and in intracellular compartments, and may either initiate the catabolism of sialoglycoconjugates or just cleave their sialic acid residues, and thereby contribute to temporal changes in their structure and functions. The current review summarizes emerging data demonstrating that mammalian neuraminidase 1, well known for its lysosomal catabolic function, is also targeted to the cell surface and assumes the previously unrecognized role as a structural and functional modulator of cellular receptors.", "author" : [ { "dropping-particle" : "V", "family" : "Pshezhetsky", "given" : "A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ashmarina", "given" : "L I", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochemistry (Moscow)", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "736-745", "title" : "Desialylation of surface receptors as a new dimension in cell signaling", "type" : "article-journal", "volume" : "78" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(15)", "plainTextFormattedCitation" : "(15)", "previouslyFormattedCitation" : "¹⁵" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(15) (See Methods). Three exemplar systems were employed to fully evaluate platelet-mediated secretion of TPO by HepG2 cells (see Methods and Supplementary Information): 1) TPO secretion from HepG2 cells incubated with artificially aged platelets (anti-TPO + desialylated PL) (~99% desialylation, see Supplementary Information). 2) TPO secretion from HepG2 cells incubated with human platelets with naturally occurring levels of sialyation (anti-TPO + sialylated PL) (~8-9% desialylation, see Supplementary Information). 3) Inherent TPO secretion from HepG2 cells (anti-TPO). An additional control measurement involving a surface functionalized with anti-IgG was also performed to characterize the degree of non-specific binding (anti-IgG). While HepG2 cells inherently secrete TPO (Figure 3f,i), the expression of TPO is consistently higher for HegG2 cells challenged with desialylated platelets (Figure 3b, f, g). Not only do we observe upregulation of TPO upon exposure to desialylated platelets, but we also see that TPO secretion is highly consistent between individual HepG2 cells (total of 30 cells). In contrast, TPO secretion from HepG2 cells challenged with naturally occurring levels of desialylated platelets (Figure 3f,h) displayed wide heterogeneity. We believe this reflects the heterogeneity of platelet age. Specifically, we find only 8 – 9% of platelets in the extracted sample are desialylated (see Supplementary Information) and are thus able to upregulate TPO expression in the HepG2 cells. These measurements agree with the recent theory of TPO regulation which states that TPO levels are directly dependent on the concentration of circulating desialylated platelets. This validation highlights how our system can provide a simple, yet powerful approach for studying complex physiological systems in vitro at the single-cell resolution. We have presented a real-time and label-free single-cell signalling analysis method based on photonic crystal resonant surfaces. The high spatial resolution of the method, coupled with the large field of view (whose dimensions are ultimately limited by the optical objective integrated in the system) and molecular specificity of the assay, enables high throughput measurements of the dynamics of protein secretion from single-cells within a cell population. These capabilities were used to examine and quantify the dynamics of cell-signalling heterogeneity within cell populations in vitro and in a timely manner appropriate for clinical applications. In particular, we were able to quantify TPO release at single cell resolution and were able to show that TPO levels are directly dependent on the concentration of circulating desialylated platelets. Given its ease of use, simple computing implementation and compatibility with standard inverted microscopes and other imaging techniques such as phase-contrast microscopy, we anticipate that PCRS the method will transform single-cell protein expression analysis from an experts-only technique into a broadly accessible single-cell signalling imaging methodology.MethodsPhotonic crystal resonant imagining surfaceThe imaging surface consists of a grating etched into a 150 nm thick silicon nitride (Si3N4) film on glass and is fabricated using electron beam lithography and reactive ion etching, which allows for multiple re-uses after cleaning. For more detail, refer to our previous workADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1364/OPTICA.4.000229", "abstract" : "Advanced biomedical diagnostic technologies fulfill an important role in improving health and well-being in society. A large number of excellent technologies have already been introduced and have given rise to the &#x201C;lab-on-a-chip&#x201D; paradigm. Most of these technologies, however, require additional instrumentation for interfacing and readout, so they are often confined to the laboratory and are not suitable for use in the field or in wider clinical practice. Other technologies require a light coupling element, such as a grating coupler or a fiber coupler, which complicates packaging. Here, we introduce a novel biosensor based on a chirped guided-mode resonant grating. The chirped grating combines the sensing function with the readout function by translating spectral information into spatial information that is easily read out with a simple CMOS camera. We demonstrate a refractive index sensitivity of 137 nm/RIU and an extrapolated limit of detection of 267 pM for the specific binding of an immunoglobulin G antibody. The chirped guided-mode resonance approach introduces a new degree of freedom for sensing biomedical information that combines high sensitivity with autonomous operation. We estimate that the cost of components is U.S. $10 or less when mass manufactured, so the technology has the potential to truly transform point-of-care applications. ", "author" : [ { "dropping-particle" : "", "family" : "Triggs", "given" : "Graham J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "Yue", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Reardon", "given" : "Christopher P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fischer", "given" : "Matthias", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Evans", "given" : "Gareth J O", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Krauss", "given" : "Thomas F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Optica", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2017" ] ] }, "page" : "229-234", "publisher" : "OSA", "title" : "Chirped guided-mode resonance biosensor", "type" : "article-journal", "volume" : "4" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1109/JPHOT.2015.2435699", "ISSN" : "19430655", "PMID" : "26356353", "abstract" : "By depositing a resolution test pattern on top of a Si3N4 photonic crystal resonant surface, we have measured the dependence of spatial resolution on refractive index contrast \u0394n. Our experimental results and finite-difference time-domain (FDTD) simulations at different refractive index contrasts show that the spatial resolution of our device reduces with reduced contrast, which is an important consideration in biosensing, where the contrast may be of order 10-2. We also compare 1-D and 2-D gratings, taking into account different incidence polarizations, leading to a better understanding of the excitation and propagation of the resonant modes in these structures, as well as how this contributes to the spatial resolution. At \u0394n 0.077, we observe resolutions of 2 and 6 \u03bcm parallel to and perpendicular to the grooves of a 1-D grating, respectively, and show that for polarized illumination of a 2-D grating, resolution remains asymmetrical. Illumination of a 2-D grating at 450 results in symmetric resolution. At very low index contrast, the resolution worsens dramatically, particularly for \u0394n <; 0.01, where we observe a resolution exceeding 10 \u03bcm for our device. In addition, we measure a reduction in the resonance linewidth as the index contrast becomes lower, corresponding to a longer resonant mode propagation length in the structure and contributing to the change in spatial resolution.", "author" : [ { "dropping-particle" : "", "family" : "Triggs", "given" : "G. J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fischer", "given" : "M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stellinga", "given" : "D.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scullion", "given" : "M. G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Evans", "given" : "G. J.O.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Krauss", "given" : "T. F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "IEEE Photonics Journal", "id" : "ITEM-2", "issue" : "3", "issued" : { "date-parts" : [ [ "2015" ] ] }, "title" : "Spatial resolution and refractive index contrast of resonant photonic crystal surfaces for biosensing", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(10, 16)", "plainTextFormattedCitation" : "(10, 16)", "previouslyFormattedCitation" : "^10,16" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(10, 16). First, the Si3N4 is cleaned in a piranha solution (1:3 hydrogen peroxide: sulfuric acid), rinsed in acetone and isopropanol, and dried with nitrogen. Next, the e-beam resist (ARP-09, AllResist GmbH) is spin-coated for 60 s at 3000 rpm, and soft baked on a hot plate at 180°C for 10 min. For charge dissipation during e-beam exposure, a thin film of aluminium (～ 20 nm) is deposited on top of the resist using a thermal evaporator (HEX, Mantis). The e-beam system (Voyager, Raith GmbH) exposes the resist with a base dose of 130 μC/cm2, after which it is developed for 2 min in Xylene, then rinsed with isopropanol and dried in N2. A sensor area of 2 x 2 mm is exposed per device, consisting of a grating with period?of 555?nm?and a fill factor?of 80% to obtain a resonance wave centred around 840 nm wavelength. To transfer the grating into the Si3N4, it is etched with a CHF3:O2 gas mixture at 29:1 sccm, an RF power of 40 W, and a chamber pressure of 1.9 e?1 mbar, over 7 min etch time. Finally, to strip the remaining resist, the sample is sonicated in 1165 solvent (MicroChem) for 3 min, followed by a rinse in acetone and isopropanol, following a drying step with N2.Hyperspectral imaging measuring setupA resonance image is formed by taking a sequence of bright field images, each at a different illumination wavelength which is achieved by illuminating through a tuneable filter (hyperspectral imaging). The resulting hyperspectral cube contains the spectrum from every pixel in the field of view. By analysing the intensity values of each pixel, the resonance wavelength for each pixel can be determined. We fit the measured data from each pixel to a Fano resonance curve to accurately obtain the resonance wavelength with pm precision. Plotting the resonance wavelength of each pixel in the array then gives the resonance image. We repeat this process cyclically over time, being limited to a rate of 2.5 min per image by instrumental limitations (we note this can be improved to sub 30 seg acquisition rates under appropriate conditions).?The camera employed here is a CoolSnap Myo (Photometrics), and the objective lens is an Olympus NeoDplan 10x (NA = 0.25) which is the component that ultimately limits the field-of-view. The camera pixel size is 4.54 μm, and after magnification, each image pixel images a size of 0.925 μm. To provide some flexibility in matching the illumination wavelength to the resonance wavelength of the fabricated grating, we use a broadband laser (SM30, Leukos), combined with a custom-built grating-based monochromator to select a single illumination wavelength with a spectral width of 0.6 nm (0.25 nm wavelength step). To further improve the resolution of the images we interpolate the values between pixels down to low pm values. Images were captured using LabView, and image analysis and curve fitting were performed using MatLab. The pixel fraction histogram to set the 1 nm threshold is obtained by dividing the total number of resonant pixels at a specific wavelength against the total number of pixels in the region of interest. We also incorporated a fluidic well for cell culturing made from acrylic, which is attached to the sensor chip to allow exposure to various analytes, while encapsulating the whole device setup inside an incubator set at 37°C.Specific surface functionalisation towards TPO proteinsSi3N4 sensor chips are carefully cleaned prior to use, consisting of a 10 min cleaning step in an ultraviolet?ozone cleaner., followed by placing in solutions of Hellmanex III (2%) and ultrapure Milli-Q water (twice) and sonication in each bath for 10 min. The sensors are then dried with N2 gas and returned to the ultraviolet?ozone cleaner for 30 min, following an exposure to 15% nitric acid solution to reveal the oxide groups on the Si3N4?surface. Finally, the clean and hydroxyl-activated sensors are immediately transferred in a 3 % v/v aminopropyltriethoxy silane (APTES) solution in ethanol and left overnight. Finally, the chips are dried with N2 gas and a curing step is performed at 110?°C for 1?hour., then loaded in the acrylic mount of the measuring setup (temperature controlled at 37°C) and injection of Milli-Q water (see Supplementary Information). Oxidized 2% (20 mg/mL) dextran solution (Dextran T40 (40 kDa), 30 mM NaIO4, Sigma Aldrich) is sequentially injected and left for 90 minADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1039/b800297e", "ISBN" : "1473-0197", "ISSN" : "1473-0197", "PMID" : "18584097", "abstract" : "The prognosis for patients suffering from cardiovascular and many other diseases can be substantially improved if diagnosed at an early stage. High performance diagnostic testing using disposable microfluidic chips can provide a platform for realizing this vision. Amic AB (Uppsala, Sweden) has developed a new microfluidic test chip for sandwich immunoassays fabricated by injection molding of the cycloolefin-copolymer Zeonor. A highly ordered array of micropillars within the fluidic chip distributes the sample solution by capillary action. Since wetting of the pillar array surface is the only driving force for liquid distribution precise control of the surface chemistry is crucial. In this work we demonstrate a novel protocol for surface hydrophilization and antibody immobilization on cycloolefin-copolymer test chips, based on direct silanisation of the thermoplastic substrate. Dextran is subsequently covalently coupled to amino groups, thus providing a coating with a low contact angle suitable for antibody immobilization. The contact angle of dextran coated chips is stable for at least two months, which enables production of large batches that can be stored for extended periods of time. We demonstrate the utility of the presented platform and surface chemistry in a C-reactive protein assay with a detection limit of 2.6 ng ml(-1), a dynamic range of 10(2) and a coefficient of variance of 15%.", "author" : [ { "dropping-particle" : "", "family" : "J\u00f6nsson", "given" : "Christina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Aronsson", "given" : "Magnus", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rundstr\u00f6m", "given" : "Gerd", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pettersson", "given" : "Christer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mendel-Hartvig", "given" : "Ib", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bakker", "given" : "Jimmy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Martinsson", "given" : "Erik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Liedberg", "given" : "Bo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "MacCraith", "given" : "Brian", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ohman", "given" : "Ove", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Melin", "given" : "Jonas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Lab on a chip", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2008" ] ] }, "page" : "1191-7", "title" : "Silane-dextran chemistry on lateral flow polymer chips for immunoassays.", "type" : "article-journal", "volume" : "8" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(17)", "plainTextFormattedCitation" : "(17)", "previouslyFormattedCitation" : "¹⁷" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(17), rinsed in Milli-Q water, further oxidized in 30 mM NaIO4 for 90 min and rinsed again in Milli-Q water (see Supplementary Information). The surface is then rinsed in phosphate-buffered saline (PBS, pH 7.4) and E.coli derived animal-free recombinant human TPO (PeproTech) antibodies are thereafter injected at 50 μg/mL in PBS (pH 7.4) and left for 60 min. To prevent non-specific binding from other biomolecules expressed from either BHK or HepG2 cell lines, casein-blocking agent is incorporated in the assay. We found an optimal concentration of 0.35x and 0.5x (in PBS pH 7.4) for BHK and HepG2 cell lines, respectively (see Supplementary information), which was incubated for 45 min to complete the sensor functionalisation. Once reached this point, the sensor is washed with cell culture media (DMEM supplemented with 10% FBS, 1% Pen-Strep and 25 mM Hepes, Gibco) prior to introducing the live cells. Cell adsorption kinetics modellingReal-time expression of signalling molecules from single cells and their binding to the capture molecules of the functionalised sensor surface was modelled as a Langmuir adsorption isotherm by assuming both processes as the same, global adsorption process. The equation to fit the secretion area of each cell per time unit is:Secretion area=N0κ*t1+κ*t (Equation 1)with N0 the offset of the area covered by the binding of signalling molecules in ?m2, κ the Langmuir adsorption rate (?m2/h) and t the time in h. A least-squares regression analysis was used to find the best fit for our dataset, which yields a coefficient of determination R2 of 0.94 ± 0.04 for the 30 BHK-TPO cell population under study.BHK-TPO and HepG2 cell lines cultureBHK-TPO and HepG2 cells were cultured at 37 °C in a 5% CO2 incubator in DMEM supplemented with 10% FBS, 1% Pen-Strep and 25 mM Hepes. Both BHK-TPO and HepG2 sub-confluent cultures were kept at 2-4x10,000 cells/cm2. Cell monolayers were passaged following a rinse with 1x PBS (twice) and pre-warmed (37°C) TrypLE Express (Gibco) solution to cover the bottom of the flask; incubated for 5 and 10 minutes, respectively. Once cells detached TrypLE Express was neutralized by adding 2x volume of complete growth medium.Isolation of human platelets from whole blood and platelet countVenous blood was collected from healthy volunteers and platelets were prepared following the protocol described inADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/S0076-6879(07)26006-9", "ISBN" : "9780123739247", "ISSN" : "00766879", "PMID" : "17697882", "abstract" : "Integrin-mediated cellular events affect all cell types and functions, in physiological as well as pathological settings. Blood platelets, because of their unique nature, have proven to be a powerful cell model with which to study the adhesive and signaling properties of integrins. The characterization of the structural and molecular mechanisms regulating the main platelet integrin, \u03b1IIb\u03b23, has provided some essential clues as to how integrins are regulated in general. The present chapter details the various protocols and reagents currently in use in our laboratory to study \u03b1IIb\u03b23 adhesive responses and signaling in both human and murine cell models. \u00a9 2007 Elsevier Inc. All rights reserved.", "author" : [ { "dropping-particle" : "", "family" : "Pr\u00e9vost", "given" : "Nicolas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kato", "given" : "Hisashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bodin", "given" : "Laurent", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shattil", "given" : "Sanford J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Methods in Enzymology", "id" : "ITEM-1", "issue" : "07", "issued" : { "date-parts" : [ [ "2007" ] ] }, "page" : "103-115", "title" : "Platelet Integrin Adhesive Functions and Signaling", "type" : "article-journal", "volume" : "426" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(18)", "plainTextFormattedCitation" : "(18)", "previouslyFormattedCitation" : "¹⁸" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(18). In short, using a 19-G butterfly needle, venous blood is taken into vacutainers and anticoagulated 5:1 with acid-citrate-dextrose (ACD) (65 mM trisodium citrate, 70 mM citric acid, 100mM dextrose, pH 4.4). Platelet-rich plasma (PRP) is obtained by centrifugation at 100x g for 20 min at room temperature. Platelets are then resuspended in wash buffer (150 mM NaCl, 20mM HEPES, pH 6.5; both from Sigma Aldrich) and centrifuged at 220x g for 10 min at room temperature in the presence of 1 U/mL apyrase (Sigma Aldrich) and 1 mM prostaglandin E1 (PGE1, Sigma Aldrich). Finally, the platelet pellet is resuspended in Walsh’s buffer (137mM NaCl, 20mMPIPES, 5.6 mM dextrose, 1 g/liter BSA, 1 mM MgCl2, 2.7 mM KCl, 3.3 mM NaH2PO4, pH 7.4; all from Sigma Aldrich). Prior to any experimental procedure, platelets are left at room temperature for 30 min; after 30min, most of the PGE1 has become inactive. Flow cytometry was employed to count the number of platelets per millilitre (see Supplementary Information).Platelet desialylation Human platelets were treated with α2–3,6,8 neuraminidase from C. perfringens (New England Biolabs) to remove terminal sialic acidADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1073/pnas.1707662114", "ISSN" : "1091-6490", "PMID" : "28716912", "abstract" : "Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1 -/- ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1 -/- mice show reduced levels of \u03b1-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1 -/- platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1 -/- platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1 -/- platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.", "author" : [ { "dropping-particle" : "", "family" : "Li", "given" : "Yun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fu", "given" : "Jianxin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ling", "given" : "Yun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yago", "given" : "Tadayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "McDaniel", "given" : "J Michael", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Song", "given" : "Jianhua", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bai", "given" : "Xia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kondo", "given" : "Yuji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Qin", "given" : "Yannan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hoover", "given" : "Christopher", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "McGee", "given" : "Samuel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shao", "given" : "Bojing", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Liu", "given" : "Zhenghui", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sonon", "given" : "Roberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Azadi", "given" : "Parastoo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Marth", "given" : "Jamey D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "McEver", "given" : "Rodger P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruan", "given" : "Changgeng", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xia", "given" : "Lijun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Proceedings of the National Academy of Sciences of the United States of America", "id" : "ITEM-1", "issue" : "31", "issued" : { "date-parts" : [ [ "2017", "8", "1" ] ] }, "page" : "8360-8365", "publisher" : "National Academy of Sciences", "title" : "Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells.", "type" : "article-journal", "volume" : "114" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(19)", "plainTextFormattedCitation" : "(19)", "previouslyFormattedCitation" : "¹⁹" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(19). Isolated platelets in Walsh’s buffer (~1.5x109 platelets/mL) were incubated with 2.5 mU of α2–3,6,8 neuraminidase for 15 minutes at 37 °C. Desialylation was confirmed by lectin binding (biotinylated version of sambucus nigra (Vector laboratories Ltd)) via flow cytometry (see Supplementary Information). Acknowledgements The research was supported by the MRC Discovery Grant MC_PC_15073 and by the EPSRC Programme Grant EP/P030017/1. The dataset associated with this research will be available online from the University of York Data Catalogue at DOI 10.15124/dffb9f91-cd1e-490e-8813-da88da18f16b. We thank Oliver Herd for his assistance in optimizing the different cell lines culture and in performing qPCR control experiments and the Imaging and Cytometry Technology Facility in the Department of Biology, University of York.Author contributionsJ.J.C. designed the structures, fabricated the samples, developed the measuring method and performed the calculations and measurements. S.J. and T.F.K. conceived the idea and supervised the project. I.S.H. and M. C. developed the idea of quantifying heterogeneity in the different cell lines. All authors contributed to the writing of the peting financial interestsThe authors declare no competing financial interests.References ADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY 1. Mukherjee P, Mani S (2013) Methodologies to decipher the cell secretome. Biochim Biophys Acta - Proteins Proteomics 1834(11):2226–2232.2. Altschuler SJ, Wu LF (2010) Cellular Heterogeneity: Do Differences Make a Difference? Cell 141(4):559–563.3. Shirasaki Y, et al. (2014) Real-time single-cell imaging of protein secretion. Sci Rep 4. doi:10.1038/srep04736.4. Li X, et al. (2018) Label-Free Optofluidic Nanobiosensor Enables Real-Time Analysis of Single-Cell Cytokine Secretion. Small 1800698:1–11.5. McDonald MP, et al. (2018) Visualizing Single-Cell Secretion Dynamics with Single-Protein Sensitivity. Nano Lett 18(1):513–519.6. Pitruzzello G, Krauss TF (2018) Photonic crystal resonances for sensing and imaging. J Opt. doi:10.1088/2040-8986/aac75b.7. Triggs GJ, et al. (2015) Spatial resolution and refractive index contrast of resonant photonic crystal surfaces for biosensing. IEEE Photonics J 7(3). doi:10.1109/JPHOT.2015.2435699.8. Estevez MC, Alvarez M, Lechuga LM (2012) Integrated optical devices for lab-on-a-chip biosensing applications. Laser Photon Rev 6(4):463–487.9. Linden HM, Kaushansky K (2002) The Glycan Domain of Thrombopoietin (TPO) Acts intrans to Enhance Secretion of the Hormone and Other Cytokines . J Biol Chem 277(38):35240–35247.10. Triggs GJ, et al. (2015) Spatial resolution and refractive index contrast of resonant photonic crystal surfaces for biosensing. IEEE Photonics J 7(3). doi:10.1109/JPHOT.2015.2435699.11. Kuter DJ, Rosenberg RD (1995) The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood 85(10):2720 LP-2730.12. Fielder PJ, et al. (1996) Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets. Blood 87(6):2154 LP-2161.13. Christoph E, Markus L, Horst F, Stephan S (2001) Endogenous thrombopoietin serum levels during multicycle chemotherapy. Br J Haematol 105(3):832–838.14. Grozovsky R, et al. (2015) The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling. Nat Med 21(1):47–54.15. Pshezhetsky A V, Ashmarina LI (2013) Desialylation of surface receptors as a new dimension in cell signaling. Biochem 78(7):736–745.16. Triggs GJ, et al. (2017) Chirped guided-mode resonance biosensor. Optica 4(2):229–234.17. J?nsson C, et al. (2008) Silane-dextran chemistry on lateral flow polymer chips for immunoassays. Lab Chip 8(7):1191–7.18. Prévost N, Kato H, Bodin L, Shattil SJ (2007) Platelet Integrin Adhesive Functions and Signaling. Methods Enzymol 426(07):103–115.19. Li Y, et al. (2017) Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells. Proc Natl Acad Sci U S A 114(31):8360–8365.Figure LegendsFigure 1. Photonic crystal resonant imaging protocol for monitoring TPO secretion from a single BHK-TPO cell. a Comparison between a photonic crystal resonant imaging hyperspectral image (top) and a phase-contrast microscope image (bottom) of a single BHK cell attached to a PCRS. Higher resonance wavelength shifts observed using PCRS are related to higher refractive index values, originating from the presence of both the cell and the secreted biomolecules. Secreted molecules are transparent and cannot be resolved by phase contrast microscopy. b Dextran molecules are employed to create a 3D network of antibodies. Casein protein is employed as a blocking agent to prevent non-specific binding from other proteins expressed by the cells. The secreted cytokines (i.e. TPO) from attached cells diffuse through the 3D network to specifically bind to the antibodies immobilized throughout the dextran network. The amount of deposited casein protein is optimised to maximise the signal-to-noise ratio of the detection system. c Gaussian distribution of the fraction of resonant pixels over the defined region of interest prior to BHK-TPO immobilization. Here the width of the Gaussian distribution was ± 0.5 nm and the mean was set to 0 nm. d The region of interest (55 x 40 ?m), with a wavelength uniformity of ?? ± 0.5 nm. e No content is observed for the chosen threshold value of ???1 nm, indicating that no cell is present. f Upon attachment of the cell to the surface, the distribution of the pixel probability of the resonance wavelength over the defined region of interest increases to -0.2<???2.2 nm, indicating the presence of biomolecular content associated to the presence of both BHK-TPO and secreted TPO. g A hyperspectral PCRS image reveals the adhesion of a BHK-TPO cell to the PCRS, whose high concentration of CAMs located in the inner region are translated into a higher refractive index content area. h By setting a threshold of 1 nm from the central resonance wavelength, a contour plot is obtained representing the area defined by the adhered BHK-TPO cell. i Over time, this secretion area increases as TPO molecules are secreted from the cell and bind to the surface immobilised antibodies, therefore locally increasing the refractive index around the BHK-TPO area. j The secretion of TPO is then monitored over time, and a Langmuir adsorption distribution is fitted to the data to model the secretion from the BHK-TPO cell accounting for the area covered by the adhered cell.Figure 2. Single-cell dynamics analysis of a 30 BHK-TPO cell population. a-d Hyperspectral contour plots (at a threshold of 1 nm above the average resonance wavelength) at 0, 8, 15 and 23 min, respectively of a 160 x 210 ?m region of interest where BHK-TPO cells attach to the functionalised PCRS. e Individual traces of the real-time secretion area coverage of a 30 cell BHK-TPO population on a TPO specific functionalized surface. The robustness and the versality of our method was further demonstrated by challening the PCRS with two different 30 cell population control systems: f firstly with a system in where the surface was rendered IgG selective (the fluctuation over time is attributed to the accumulation of non-specific interactions between the secreted TPO and immobilised IgG antibodies), and g secondly with a BHK-TPO population treated with the protein transport inhibitor brefeldin A. h The real-time response from the three systems over 130 min yielded notable and quantitative differences. The direct system (BHK-TPO with a TPO selective PCRS functionalization, in red) exhibited an average secretion area coverage ?anti-TPO ~ 720 ?m2, while this average secretion area coverage was nearly reduced to a half in the two control systems (?anti-IgG ~ 400 ?m2 (orange), ?brefeldin A ~ 300 ?m2 (green)), indicating the specificty and robustness of both assay and extraction method. The shaded areas represent the standard deviation for each trace at that time instant. i We analyzed the heterogeneity within each 30 cell population by studying the standard deviation in secretion area coverage distribution at 120 min, which revealed a ~ 2-3 fold higher secretion area in the direct system over the two control systems. j We modelled the Langmuir rate constant associated with each system to deconvolute the influence of the inherent cell attachment area from the measurement. We obtained rates of ~ 22, 9 and 6.5 ?m2/h for the direct, non-specific and non-expressing systems, respectively.Figure 3. Levels of desialylated platelets affect TPO expression from HepG2 cells. a Real-time TPO secretion from a population of 30 HepG2 cells using hyperspectral PCRS imaging. Average TPO secretion from HepG2 cells cultured in a 1000 platelets/cell media with 99 % desialylated platelets is 1.5 times higher than the inherent TPO (see Methods and Supplementary Information). The shaded areas represent the standard deviation for each trace at that time instant. b Traces within the 30 HepG2 cell population exhibit a small standard deviation in the area of secretion of ?anti-TPO,-sia ~ 320 ?m2. c In contrast, the HepG2 cell population cultured in a 1000 platelets/cell media with 8-9 % desialylated platelets show a standard deviation of ?TPO,+sia ~ 600 ?m2, indicating a high degree of heterogeneity due to unsynchronised and uneven TPO expression within the population with respect to the system shown in b). d-e Reference and control systems, respectively, exhibit lower TPO expression and similar heterogeneity (?anti-TPO and ?anti-IgG ~ 280 and 330 ?m2, respectively) to the system challenged with a rich desialylated platelet media. f Scatter plot of the individual secretion area from each individual cell within the population of the different systems after 180 min. The box indicates the 25th and 75th percentiles of the samples, respectively. TPO secretion from cells exposed to 99 % and 8-9 % desialylated platelets, respectively, was found to be κanti-TPO + desialylated PL 17 ?m2/h and κanti-TPO + sialylated PL 13 ?m2/h (see Supplementaty Information). g-j Cell count histograms of the secretion area for each HepG2 cell population (including the control system functionalised with an antibody against IgG). The standard deviation for each case is represented with a bar in each of the plots, which directly relates to the heterogeneity of the system. ................
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