The Experts below are selected from a list of 10929 Experts worldwide ranked by ideXlab platform
Garry P Nolan - One of the best experts on this subject based on the ideXlab platform.
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profiling myelodysplastic syndromes by Mass Cytometry demonstrates abnormal progenitor cell phenotype and differentiation
bioRxiv, 2019Co-Authors: Gregory K. Behbehani, Rachel Finck, Wendy Fantl, Nikolay Samusik, Kunju Sridhar, Peter L Greenberg, Garry P NolanAbstract:Abstract Purpose We sought to enhance the cytometric analysis of MDS by performing a pilot study of a single cell Mass Cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS. Experimental Design Twenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter Mass Cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering. Results This high-dimensional assay provided three major benefits relative to traditional Cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. Second, analysis of the stem and progenitor cell compartment (HSPCs), demonstrated aberrant expression in 21 of the 23 MDS samples, which were not detected in three samples from patients with idiopathic cytopenia of undetermined significance (ICUS). These immunophenotypically abnormal HSPCs were also the single most significant distinguishing feature between clinical risk groups. Third, unsupervised clustering of high-parameter MCM data allowed identification of abnormal differentiation patterns associated with immunophenotypically aberrant myeloid cells similar to myeloid derived suppressor cells. Conclusions These results demonstrate that high-parameter Cytometry methods that enable simultaneous analysis of all bone marrow cell types could enhance the diagnostic utility of immunophenotypic analysis in MDS. Statement of Significance High-dimensional Mass Cytometry enables high-resolution characterization of abnormal maker expression and myeloid development in MDS. This technology could enhance MDS diagnosis and therapeutic monitoring and merits further research. Statement Translational Relevance In spite of several studies suggesting the utility of flow Cytometry in the diagnosis of myelodysoplastic syndrome (MDS), this technique has not been widely adopted. We sought to enhance the utility of Cytometry in MDS by performing the first high-dimensional Mass Cytometry characterization of a cohort of MDS patients. High-dimensional Mass Cytometry allowed all bone marrow cell populations to be simultaneously analyzed enabling high-resolution characterization of abnormal maker expression and myeloid development in MDS. This approach could identify almost all previously identified aberrant surface marker expression patterns in MDS while simultaneously enabling analysis by unsupervised clustering. Additionally, this Mass Cytometry analysis approach enabled the modeling of abnormal differentiation in MDS. This technology could enhance MDS diagnosis and therapeutic monitoring and merits further research.
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Metal-isotope-tagged monoclonal antibodies for high-dimensional Mass Cytometry.
Nature Protocols, 2018Co-Authors: Matthew H. Spitzer, Wendy Fantl, Sean C Bendall, Garry P NolanAbstract:Advances in single-cell Mass Cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in Mass Cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic Mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in Cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium Mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h. The immunoassay multiplexing capacity of single-cell Mass Cytometry relies on monoclonal antibodies labeled with stable heavy-metal isotopes. Nolan et al. describe procedures for conjugating monoclonal IgG antibodies with 48 heavy-metal isotopes.
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High-resolution myogenic lineage mapping by single-cell Mass Cytometry
Nature cell biology, 2017Co-Authors: Ermelinda Porpiglia, Astraea Jager, Garry P Nolan, Sean C Bendall, Kara L Davis, Nikolay Samusik, Andrew Tri Van Ho, Benjamin D. Cosgrove, Thach Mai, Wendy FantlAbstract:Porpiglia et al. use single-cell Mass Cytometry to analyse surface markers and key myogenic transcription factors of skeletal muscle stem cells during homeostasis and repair, and identify previously unrecognized myogenic progenitor cell populations.
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Mass Cytometry single cells many features
Cell, 2016Co-Authors: Matthew H. Spitzer, Garry P NolanAbstract:Technology development in biological research often aims to either increase the number of cellular features that can be surveyed simultaneously or enhance the resolution at which such observations are possible. For decades, flow Cytometry has balanced these goals to fill a critical need by enabling the measurement of multiple features in single cells, commonly to examine complex or hierarchical cellular systems. Recently, a format for flow Cytometry has been developed that leverages the precision of Mass spectrometry. This fusion of the two technologies, termed Mass Cytometry, provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of Cytometry to evaluate complex cellular systems and processes. In this Primer, we review the current state of Mass Cytometry, providing an overview of the instrumentation, its present capabilities, and methods of data analysis, as well as thoughts on future developments and applications.
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Mass Cytometry to Decipher the Mechanism of Nongenetic Drug Resistance in Cancer
Current topics in microbiology and immunology, 2014Co-Authors: Harris G Fienberg, Garry P NolanAbstract:Nongenetic resistance has recently been described as a major impediment to effective cancer therapy. Nongenetic resistance is challenging to study since it occurs nonuniformly, even in cell lines, and can involve the interplay of multiple survival pathways. Until recently, no technology allowed measurement of large-scale alterations in survival pathways with single-cell resolution. Mass Cytometry, a flow-based technique in which the activation of up to 50 proteins can be measured simultaneously in single-cell, now provides the ability to examine nongenetic resistance on the functional level on a cell-by-cell basis. The application of Mass Cytometry, in combination with new bioinformatic techniques, will allow fundamental questions on nongenetic resistance to be addressed: Is resistance caused by selection of cells with a pre-existing survival phenotype or induction of a survival program? Which survival pathways are necessary for nongenetic resistance and how do they interact? Currently, Mass Cytometry is being used to investigate the mechanism of nongenetic resistance to TRAIL-induced apoptosis. The approaches being developed to understand resistance to TRAIL will likely be applied to elucidate the mechanisms of nongenetic resistance broadly and in the clinic.
Bernd Bodenmiller - One of the best experts on this subject based on the ideXlab platform.
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compensation of signal spillover in suspension and imaging Mass Cytometry
Cell systems, 2018Co-Authors: Stephane Chevrier, Helena L Crowell, Vito R T Zanotelli, Stefanie Engler, Mark D Robinson, Bernd BodenmillerAbstract:Summary The advent of Mass Cytometry increased the number of parameters measured at the single-cell level while decreasing the extent of crosstalk between channels relative to dye-based flow Cytometry. Although reduced, spillover still exists in Mass Cytometry data, and minimizing its effect requires considerable expert knowledge and substantial experimental effort. Here, we describe a novel bead-based compensation workflow and R-based software that estimates and corrects for interference between channels. We performed an in-depth characterization of the spillover properties in Mass Cytometry, including limitations defined by the linear range of the Mass cytometer and the reproducibility of the spillover over time and across machines. We demonstrated the utility of our method in suspension and imaging Mass Cytometry. To conclude, our approach greatly simplifies the development of new antibody panels, increases flexibility for antibody-metal pairing, opens the way to using less pure isotopes, and improves overall data quality, thereby reducing the risk of reporting cell phenotype artifacts.
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Ruthenium counterstaining for imaging Mass Cytometry.
The Journal of pathology, 2018Co-Authors: Raul Catena, Luis M. Montuenga, Bernd BodenmillerAbstract:Imaging Mass Cytometry is a novel imaging modality that enables simultaneous antibody-based detection of >40 epitopes and molecules in tissue sections at subcellular resolution by the use of isotopically pure metal tags. Essential for any imaging approach in which antigen detection is performed is counterstaining, which reveals the overall structure of the tissue. Counterstaining is necessary because antigens of interest are often present in only a small subset of cells, and the rest of the tissue structures are not visible. As most biological tissues are nearly transparent or non-fluorescent, chromogenic reagents such as haematoxylin (for immunohistochemistry) or fluorescent dyes such as 4',6-diamidino-2-phenylindole (which stains nuclei for epifluorescence and confocal microscopy) are utilized. Here, we describe a metal-based counterstain for imaging Mass Cytometry based on simple oxidation and subsequent covalent binding of the tissue components to ruthenium tetroxide (RuO4 ). RuO4 counterstaining reveals general tissue structure both in areas with high cell content and in stromal areas with low cellularity and fibrous or hyaline material in a manner analogous to haematoxylin in immunohistochemical counterstaining or eosin or other anionic dyes in conventional histology. Our new counterstain approach is applicable to any metal-based imaging technique, and will facilitate the adaptation of imaging Mass Cytometry for routine applications in clinical and research laboratories. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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CellCycleTRACER accounts for cell cycle and volume in Mass Cytometry data.
Nature communications, 2018Co-Authors: Maria Anna Rapsomaniki, Bernd Bodenmiller, Xiao-kang Lun, Stefan Woerner, Marco Laumanns, María Rodríguez MartínezAbstract:Recent studies have shown that cell cycle and cell volume are confounding factors when studying biological phenomena in single cells. Here we present a combined experimental and computational method, CellCycleTRACER, to account for these factors in Mass Cytometry data. CellCycleTRACER is applied to Mass Cytometry data collected on three different cell types during a TNFα stimulation time-course. CellCycleTRACER reveals signaling relationships and cell heterogeneity that were otherwise masked.
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channel crosstalk correction in suspension and imaging Mass Cytometry
bioRxiv, 2017Co-Authors: Stephane Chevrier, Helena L Crowell, Vito R T Zanotelli, Stefanie Engler, Mark D Robinson, Bernd BodenmillerAbstract:Mass Cytometry enables simultaneous analysis of over 40 proteins and their modifications in single cells through use of metal-tagged antibodies. Compared to fluorescent dyes, the use of pure metal isotopes strongly reduces spectral overlap among measurement channels. Crosstalk still exists, however, caused by isotopic impurity, oxide formation, and Mass cytometer properties. Spillover effects can be minimized, but not avoided, by following a set of constraining rules when designing an antibody panel. Generation of such low crosstalk panels requires considerable expert knowledge, knowledge of the abundance of each marker and substantial experimental effort. Here we describe a novel bead-based compensation workflow that includes R-based software and a web tool, which enables correction for interference between channels. We demonstrate utility in suspension Mass Cytometry and show how this approach can be applied to imaging Mass Cytometry. Our approach greatly simplifies the development of new antibody panels, increases flexibility for antibody-metal pairing, improves overall data quality, thereby reducing the risk of reporting cell phenotype and function artifacts, and greatly facilitates analysis of complex samples for which antigen abundances are unknown.
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enhanced multiplexing in Mass Cytometry using osmium and ruthenium tetroxide species
Cytometry Part A, 2016Co-Authors: Raul Catena, Nevena Zivanovic, Alaz Ozcan, Bernd BodenmillerAbstract:Mass Cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in Mass Cytometry, called Mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO4 and RuO4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO4 and RuO4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO4 and RuO4 were used in a 20-sample barcoding protocol together with palladium isotopes. As Mass channels occupied by osmium and ruthenium are not used for antibody detection the number of Masses effectively utilized in a single experiment is expanded. OsO4 and RuO4 can therefore be used as MCB reagents for a wide range of Mass Cytometry workflows. © 2016 International Society for Advancement of Cytometry.
Sean C Bendall - One of the best experts on this subject based on the ideXlab platform.
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Immune monitoring using Mass Cytometry and related high-dimensional imaging approaches
Nature Reviews Rheumatology, 2020Co-Authors: Felix J. Hartmann, Sean C BendallAbstract:Immune monitoring of human cells using systems immunology approaches has the potential to produce new insights into pathological processes and therapeutic opportunities for rheumatic disease research. Proteomic approaches that use elemental (heavy metal) reporter ions, such as Mass Cytometry and high-dimensional imaging techniques, might be of value for the study of a wide variety of clinical samples. Mass Cytometry enables in-depth analysis of the phenotype and functional state of immune cells at the single-cell level. High-dimensional imaging techniques use concepts analogous to Mass Cytometry to image cells in their histological context, providing spatial and cell–cell interaction information. A combination of these technologies with data-driven analytical approaches can give predictive insights into disease mechanisms for rheumatic diseases. Single-cell proteomic techniques that use elemental (heavy metal) reporter ions increase the number of parameters that can be studied at once in whole tissues. This Review discusses the practical aspects of using such technologies in rheumatic disease research. The cellular complexity and functional diversity of the human immune system necessitate the use of high-dimensional single-cell tools to uncover its role in multifaceted diseases such as rheumatic diseases, as well as other autoimmune and inflammatory disorders. Proteomic technologies that use elemental (heavy metal) reporter ions, such as Mass Cytometry (also known as CyTOF) and analogous high-dimensional imaging approaches (including multiplexed ion beam imaging (MIBI) and imaging Mass Cytometry (IMC)), have been developed from their low-dimensional counterparts, flow Cytometry and immunohistochemistry, to meet this need. A growing number of studies have been published that use these technologies to identify functional biomarkers and therapeutic targets in rheumatic diseases, but the full potential of their application to rheumatic disease research has yet to be fulfilled. This Review introduces the underlying technologies for high-dimensional immune monitoring and discusses aspects necessary for their successful implementation, including study design principles, analytical tools and future developments for the field of rheumatology.
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immune monitoring using Mass Cytometry and related high dimensional imaging approaches
Nature Reviews Rheumatology, 2020Co-Authors: Felix J. Hartmann, Sean C BendallAbstract:The cellular complexity and functional diversity of the human immune system necessitate the use of high-dimensional single-cell tools to uncover its role in multifaceted diseases such as rheumatic diseases, as well as other autoimmune and inflammatory disorders. Proteomic technologies that use elemental (heavy metal) reporter ions, such as Mass Cytometry (also known as CyTOF) and analogous high-dimensional imaging approaches (including multiplexed ion beam imaging (MIBI) and imaging Mass Cytometry (IMC)), have been developed from their low-dimensional counterparts, flow Cytometry and immunohistochemistry, to meet this need. A growing number of studies have been published that use these technologies to identify functional biomarkers and therapeutic targets in rheumatic diseases, but the full potential of their application to rheumatic disease research has yet to be fulfilled. This Review introduces the underlying technologies for high-dimensional immune monitoring and discusses aspects necessary for their successful implementation, including study design principles, analytical tools and future developments for the field of rheumatology.
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Metal-isotope-tagged monoclonal antibodies for high-dimensional Mass Cytometry.
Nature Protocols, 2018Co-Authors: Matthew H. Spitzer, Wendy Fantl, Sean C Bendall, Garry P NolanAbstract:Advances in single-cell Mass Cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in Mass Cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic Mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in Cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium Mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h. The immunoassay multiplexing capacity of single-cell Mass Cytometry relies on monoclonal antibodies labeled with stable heavy-metal isotopes. Nolan et al. describe procedures for conjugating monoclonal IgG antibodies with 48 heavy-metal isotopes.
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High-resolution myogenic lineage mapping by single-cell Mass Cytometry
Nature cell biology, 2017Co-Authors: Ermelinda Porpiglia, Astraea Jager, Garry P Nolan, Sean C Bendall, Kara L Davis, Nikolay Samusik, Andrew Tri Van Ho, Benjamin D. Cosgrove, Thach Mai, Wendy FantlAbstract:Porpiglia et al. use single-cell Mass Cytometry to analyse surface markers and key myogenic transcription factors of skeletal muscle stem cells during homeostasis and repair, and identify previously unrecognized myogenic progenitor cell populations.
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Normalization of Mass Cytometry data with bead standards
Cytometry Part A, 2013Co-Authors: Rachel Finck, Astraea Jager, Karen Sachs, Wendy Fantl, Erin F Simonds, Smita Krishnaswamy, Garry P Nolan, Sean C BendallAbstract:Mass Cytometry uses atomic Mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of Mass Cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of Mass Cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the Mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold.
Wendy Fantl - One of the best experts on this subject based on the ideXlab platform.
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profiling myelodysplastic syndromes by Mass Cytometry demonstrates abnormal progenitor cell phenotype and differentiation
bioRxiv, 2019Co-Authors: Gregory K. Behbehani, Rachel Finck, Wendy Fantl, Nikolay Samusik, Kunju Sridhar, Peter L Greenberg, Garry P NolanAbstract:Abstract Purpose We sought to enhance the cytometric analysis of MDS by performing a pilot study of a single cell Mass Cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS. Experimental Design Twenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter Mass Cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering. Results This high-dimensional assay provided three major benefits relative to traditional Cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. Second, analysis of the stem and progenitor cell compartment (HSPCs), demonstrated aberrant expression in 21 of the 23 MDS samples, which were not detected in three samples from patients with idiopathic cytopenia of undetermined significance (ICUS). These immunophenotypically abnormal HSPCs were also the single most significant distinguishing feature between clinical risk groups. Third, unsupervised clustering of high-parameter MCM data allowed identification of abnormal differentiation patterns associated with immunophenotypically aberrant myeloid cells similar to myeloid derived suppressor cells. Conclusions These results demonstrate that high-parameter Cytometry methods that enable simultaneous analysis of all bone marrow cell types could enhance the diagnostic utility of immunophenotypic analysis in MDS. Statement of Significance High-dimensional Mass Cytometry enables high-resolution characterization of abnormal maker expression and myeloid development in MDS. This technology could enhance MDS diagnosis and therapeutic monitoring and merits further research. Statement Translational Relevance In spite of several studies suggesting the utility of flow Cytometry in the diagnosis of myelodysoplastic syndrome (MDS), this technique has not been widely adopted. We sought to enhance the utility of Cytometry in MDS by performing the first high-dimensional Mass Cytometry characterization of a cohort of MDS patients. High-dimensional Mass Cytometry allowed all bone marrow cell populations to be simultaneously analyzed enabling high-resolution characterization of abnormal maker expression and myeloid development in MDS. This approach could identify almost all previously identified aberrant surface marker expression patterns in MDS while simultaneously enabling analysis by unsupervised clustering. Additionally, this Mass Cytometry analysis approach enabled the modeling of abnormal differentiation in MDS. This technology could enhance MDS diagnosis and therapeutic monitoring and merits further research.
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Metal-isotope-tagged monoclonal antibodies for high-dimensional Mass Cytometry.
Nature Protocols, 2018Co-Authors: Matthew H. Spitzer, Wendy Fantl, Sean C Bendall, Garry P NolanAbstract:Advances in single-cell Mass Cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in Mass Cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic Mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in Cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium Mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h. The immunoassay multiplexing capacity of single-cell Mass Cytometry relies on monoclonal antibodies labeled with stable heavy-metal isotopes. Nolan et al. describe procedures for conjugating monoclonal IgG antibodies with 48 heavy-metal isotopes.
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High-resolution myogenic lineage mapping by single-cell Mass Cytometry
Nature cell biology, 2017Co-Authors: Ermelinda Porpiglia, Astraea Jager, Garry P Nolan, Sean C Bendall, Kara L Davis, Nikolay Samusik, Andrew Tri Van Ho, Benjamin D. Cosgrove, Thach Mai, Wendy FantlAbstract:Porpiglia et al. use single-cell Mass Cytometry to analyse surface markers and key myogenic transcription factors of skeletal muscle stem cells during homeostasis and repair, and identify previously unrecognized myogenic progenitor cell populations.
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single cell Mass Cytometry for analysis of immune system functional states
Current Opinion in Immunology, 2013Co-Authors: Zach Bjornson, Garry P Nolan, Wendy FantlAbstract:Mass Cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with panels of up to 45 antibodies. Each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the lanthanide series of the periodic table. Antibody panels recognize surface markers to delineate cell types simultaneously with intracellular signaling molecules to measure biological functions, such as metabolism, survival, DNA damage, cell cycle and apoptosis, to provide an overall determination of the network state of an individual cell. This review will cover the basics of Mass Cytometry as well as outline assays developed for the platform that enhance the immunologist's analytical arsenal.
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Normalization of Mass Cytometry data with bead standards
Cytometry Part A, 2013Co-Authors: Rachel Finck, Astraea Jager, Karen Sachs, Wendy Fantl, Erin F Simonds, Smita Krishnaswamy, Garry P Nolan, Sean C BendallAbstract:Mass Cytometry uses atomic Mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of Mass Cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of Mass Cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the Mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold.
Jonathan M. Irish - One of the best experts on this subject based on the ideXlab platform.
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Beyond the message: advantages of snapshot proteomics with single‐cell Mass Cytometry in solid tumors
The FEBS journal, 2019Co-Authors: Akshitkumar M. Mistry, Allison R. Greenplate, Rebecca A. Ihrie, Jonathan M. IrishAbstract:Single-cell technologies that can quantify features of individual cells within a tumor are critical for treatment strategies aiming to target cancer cells while sparing or activating beneficial cells. Given that key players in protein networks are often the primary targets of precision oncology strategies, it is imperative to transcend the nucleic acid message and read cellular actions in human solid tumors. Here, we review the advantages of multiplex, single-cell Mass Cytometry in tissue and solid tumor investigations. Mass Cytometry can quantitatively probe nearly any cellular feature or target. In discussing the ability of Mass Cytometry to reveal and characterize a broad spectrum of cell types, identify rare cells, and study functional behavior through protein signaling networks in millions of individual cells from a tumor, this review surveys publications of scientific advances in solid tumor biology made with the aid of Mass Cytometry. Advances discussed include functional identification of rare tumor and tumor-infiltrating immune cells and dissection of cellular mechanisms of immunotherapy in solid tumors and the periphery. The review concludes by highlighting ways to incorporate single-cell Mass Cytometry in solid tumor precision oncology efforts and rapidly developing Cytometry techniques for quantifying cell location and sequenced nucleic acids.
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methods for discovery and characterization of cell subsets in high dimensional Mass Cytometry data
Methods, 2015Co-Authors: Kirsten E. Diggins, Brent P Ferrell, Jonathan M. IrishAbstract:Abstract The flood of high-dimensional data resulting from Mass Cytometry experiments that measure more than 40 features of individual cells has stimulated creation of new single cell computational biology tools. These tools draw on advances in the field of machine learning to capture multi-parametric relationships and reveal cells that are easily overlooked in traditional analysis. Here, we introduce a workflow for high dimensional Mass Cytometry data that emphasizes unsupervised approaches and visualizes data in both single cell and population level views. This workflow includes three central components that are common across Mass Cytometry analysis approaches: (1) distinguishing initial populations, (2) revealing cell subsets, and (3) characterizing subset features. In the implementation described here, viSNE, SPADE, and heatmaps were used sequentially to comprehensively characterize and compare healthy and malignant human tissue samples. The use of multiple methods helps provide a comprehensive view of results, and the largely unsupervised workflow facilitates automation and helps researchers avoid missing cell populations with unusual or unexpected phenotypes. Together, these methods develop a framework for future machine learning of cell identity.
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Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.
Methods in molecular biology (Clifton N.J.), 2015Co-Authors: Nalin Leelatian, Kirsten E. Diggins, Jonathan M. IrishAbstract:Single cell Mass Cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass Cytometry experiments routinely measure 25-35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two Mass Cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a Mass Cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank ( http://www.cytobank.org/irishlab/ ).