Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum
Sean C. Bendall,Erin F. Simonds,Peng Qiu,El-ad David Amir,Peter O. Krutzik,Rachel Finck,Robert V. Bruggner,Rachel D. Melamed,Angelica Trejo,Olga Ornatsky,Robert S. Balderas,Sylvia K. Plevritis,Karen Sachs,Dana Pe'er,Scott D. Tanner,Garry P. Nolan +15 more
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TLDR
Single-cell “mass cytometry” analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.Abstract:
Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.read more
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Journal ArticleDOI
Multiplexed single-cell proteomics using SCoPE2.
Aleksandra A. Petelski,Edward Emmott,Edward Emmott,Andrew Leduc,R. Gray Huffman,Harrison Specht,David H. Perlman,David H. Perlman,Nikolai Slavov +8 more
TL;DR: The Single Cell ProtEomics (SCoPE2) protocol as mentioned in this paper uses an isobaric carrier sample to enhance peptide sequence identification with multiplexed analysis using TMT or TMTpro.
Book ChapterDOI
Single-Cell Western Blotting.
Elly Sinkala,Amy E. Herr +1 more
TL;DR: The scWestern protein assay is capable of highly specific analysis by coupling antibody-based detection with a polyacrylamide gel electrophoresis (PAGE) protein separation and photo-immobilization of all proteins to the gel allows for antibody probing and lends to the archival quality of the scWestern assay.
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The Alternative Faces of Macrophage Generate Osteoclasts
TL;DR: The role of immune-related stimuli and factors in inducing macrophages towards the osteoclastogenesis choice is highlighted and various inflammatory stimuli known to induce macrophage polarization, such as LPS or TNF-α, can alter the type of MGC obtained from RANKL-induced differentiation.
Journal ArticleDOI
Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity
Craig Gedye,Ali Hussain,Joshua Paterson,Alannah Smrke,Harleen Saini,Danylo Sirskyj,Keira Pereira,Nazleen Lobo,Jocelyn M. Stewart,Christopher Go,Jenny M. Ho,Mauricio Medrano,Elzbieta Hyatt,Julie Yuan,Stevan Lauriault,Maria Kondratyev,Twan van den Beucken,Michael A.S. Jewett,Peter B. Dirks,Cynthia J. Guidos,Jayne S. Danska,Jean C.Y. Wang,Bradly G. Wouters,Bradly G. Wouters,Benjamin G. Neel,Benjamin G. Neel,Robert Rottapel,Laurie Ailles,Laurie Ailles +28 more
TL;DR: It is demonstrated that HT-FC provides reproducible results, and the platform is used to identify cell surface antigens that are influenced by common cell preparation methods, and it is shown that multiple populations within complex samples can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies.
Journal ArticleDOI
Single cell analysis of human tissues and solid tumors with mass cytometry.
Nalin Leelatian,Deon B. Doxie,Allison R. Greenplate,Bret C. Mobley,Jonathan M. Lehman,Justine Sinnaeve,Rondi M. Kauffman,Jay A. Werkhaven,Akshitkumar M. Mistry,Kyle D. Weaver,Reid C. Thompson,Pierre P. Massion,Mary A. Hooks,Mark C. Kelley,Lola B. Chambless,Rebecca A. Ihrie,Jonathan M. Irish +16 more
TL;DR: This investigation establishes standard procedures for preparing viable single cell suspensions that preserve the cellular diversity of human tissue microenvironments.
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Journal ArticleDOI
Causal Protein-Signaling Networks Derived from Multiparameter Single-Cell Data
Karen Sachs,Karen Sachs,Karen Sachs,Omar D. Perez,Omar D. Perez,Omar D. Perez,Dana Pe'er,Dana Pe'er,Dana Pe'er,Douglas A. Lauffenburger,Douglas A. Lauffenburger,Douglas A. Lauffenburger,Garry P. Nolan,Garry P. Nolan,Garry P. Nolan +14 more
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