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  1. AU=Tyznik Aaron J.
  2. AU="Siddiqui, Fasih Sami"
  3. AU="Abt, S."
  4. AU="Sultana, Sufia"
  5. AU="Yuan, Shengwang"
  6. AU="Sedehizadeh, Saam"
  7. AU=Jeon Young-Woo
  8. AU=Narayanan Navaneeth
  9. AU=Beckwith Kyle A.
  10. AU=Anderson Robert H
  11. AU=Trindade P A
  12. AU=Gohil R
  13. AU="Zhengyi, Wu"
  14. AU=Wessels J A M
  15. AU="Porter, Lavinia"
  16. AU=Al-Mayman Sulaiman

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  1. Artikel ; Online: OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

    Konecny, Andrew J / Mage, Peter L / Tyznik, Aaron J / Prlic, Martin / Mair, Florian

    Cytometry. Part A : the journal of the International Society for Analytical Cytology

    2024  

    Abstract: We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using ... ...

    Abstract We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.
    Sprache Englisch
    Erscheinungsdatum 2024-04-18
    Erscheinungsland United States
    Dokumenttyp Journal Article
    ZDB-ID 2099868-5
    ISSN 1552-4930 ; 0196-4763 ; 1552-4922
    ISSN (online) 1552-4930
    ISSN 0196-4763 ; 1552-4922
    DOI 10.1002/cyto.a.24841
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  2. Artikel ; Online: Co-staining with Fluorescent Antibodies and Antibody-Derived Tags for Cell Sorting Prior to CITE-Seq.

    Shi, Xiaoshan / Baracho, Gisele V / Lomas, Woodrow E / Song, Hye-Won / Widmann, Stephanie J / Tyznik, Aaron J

    Methods in molecular biology (Clifton, N.J.)

    2024  Band 2779, Seite(n) 287–303

    Abstract: The paired detection of the transcriptome and proteome at single-cell resolution provides exquisite insight to immune mechanisms in health and disease. Here, we describe a detailed protocol wherein we combine cellular indexing of transcriptomes and ... ...

    Abstract The paired detection of the transcriptome and proteome at single-cell resolution provides exquisite insight to immune mechanisms in health and disease. Here, we describe a detailed protocol wherein we combine cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), a technique utilizing antibody-derived tags (ADTs) to profile mRNA and proteins simultaneously via sequencing, with fluorescence-activated cell sorting to enrich cell populations. Our protocol provides step-by-step guidance on co-staining cells with both fluorescent antibodies and ADTs simultaneously, instructions on cell sorting and an overview of the single-cell capture workflow using the BD Rhapsody™ system. This method is useful for in-depth single-cell characterization on sorted rare cell populations.
    Mesh-Begriff(e) Gene Expression Profiling/methods ; Epitopes ; Transcriptome ; Cell Separation ; Antibodies ; Single-Cell Analysis/methods
    Chemische Substanzen Epitopes ; Antibodies
    Sprache Englisch
    Erscheinungsdatum 2024-04-01
    Erscheinungsland United States
    Dokumenttyp Journal Article
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-3738-8_13
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  3. Artikel: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

    Konecny, Andrew J / Mage, Peter / Tyznik, Aaron J / Prlic, Martin / Mair, Florian

    bioRxiv : the preprint server for biology

    2023  

    Abstract: We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using ... ...

    Abstract We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in tissue biopsies and other human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.
    Sprache Englisch
    Erscheinungsdatum 2023-12-15
    Erscheinungsland United States
    Dokumenttyp Preprint
    DOI 10.1101/2023.12.14.571745
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  4. Artikel ; Online: High-Dimensional Immunophenotyping with Fluorescence-Based Cytometry: A Practical Guidebook.

    Mair, Florian / Tyznik, Aaron J

    Methods in molecular biology (Clifton, N.J.)

    2019  Band 2032, Seite(n) 1–29

    Abstract: Recent technological advances have greatly diversified the platforms that are available for high-dimensional single-cell immunophenotyping, including mass cytometry, single-cell RNA sequencing, and fluorescent-based flow cytometry. The latter is ... ...

    Abstract Recent technological advances have greatly diversified the platforms that are available for high-dimensional single-cell immunophenotyping, including mass cytometry, single-cell RNA sequencing, and fluorescent-based flow cytometry. The latter is currently the most commonly used approach, and modern instrumentation allows for the measurement of up to 30 parameters, revealing deep insights into the complexity of the immune system.Here, we provide a practical guidebook for the successful design and execution of complex fluorescence-based immunophenotyping panels. We address common misconceptions and caveats, and also discuss challenges that are associated with the quality control and analysis of these data sets.
    Mesh-Begriff(e) Flow Cytometry/methods ; Fluorescence ; Immunophenotyping/methods ; Molecular Biology/methods ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/methods
    Sprache Englisch
    Erscheinungsdatum 2019-08-08
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-9650-6_1
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  5. Artikel ; Online: Flow cytometry analysis of protein expression using antibody-derived tags followed by CITE-Seq.

    Shi, Xiaoshan / Fan, Wei / Mehrpouyan, Majid / Chen, Yu / D'Cruz, Louise M / Widmann, Stephanie J / Tyznik, Aaron J

    Cytometry. Part A : the journal of the International Society for Analytical Cytology

    2023  Band 105, Heft 1, Seite(n) 62–73

    Abstract: Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) is a single-cell phenotyping method that uses antibody-derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single- ... ...

    Abstract Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) is a single-cell phenotyping method that uses antibody-derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single-cell level. Despite the increased popularity of this technique to study cellular heterogeneity and dynamics, detailed methods on how to choose ADT markers and ensuring reagent performance in biological relevant systems prior to sequencing is not available. Here we describe a novel and easy-to-use multiplex flow proxy assay in which multiple protein markers can be measured simultaneously using a combination of ADT reagents and dye-oligo conjugates by flow cytometry. Using dye-oligo conjugates with sequences complementary to the ADT reagents, we can achieve specific binding and evaluate protein marker expression in a multiplex way. This quality control assay is useful for guiding ADT marker choice and confirming protein expression prior to sequencing. Importantly, the labeled cells can be directly isolated based on the specific fluorescence from dye-oligo conjugates using a flow cytometry cell sorter and processed for downstream single-cell multiomics. Using this streamlined workflow, we sorted natural killer cells and T cells efficiently using only ADT and dye-oligo reagents, avoiding the possibility of decreased marker resolution from co-staining cells with ADT and fluorescent antibodies. This novel workflow provides a viable option for improving ADT marker choice and cell sorting efficiency, allowing subsequent CITE-Seq.
    Mesh-Begriff(e) Flow Cytometry/methods ; Epitopes ; Antibodies ; Cell Separation/methods ; T-Lymphocytes ; Antigens ; Single-Cell Analysis
    Chemische Substanzen Epitopes ; Antibodies ; Antigens
    Sprache Englisch
    Erscheinungsdatum 2023-09-29
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2099868-5
    ISSN 1552-4930 ; 0196-4763 ; 1552-4922
    ISSN (online) 1552-4930
    ISSN 0196-4763 ; 1552-4922
    DOI 10.1002/cyto.a.24792
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  6. Artikel ; Online: Functional phenotyping of circulating human cytotoxic T cells and NK cells using a 16-color flow cytometry panel.

    Baracho, Gisele V / Kara, Nihan / Rigaud, Stephanie / Lo, Evelyn / Widmann, Stephanie J / Tyznik, Aaron J

    STAR protocols

    2021  Band 3, Heft 1, Seite(n) 101069

    Abstract: Cytotoxic T lymphocytes and natural killer (NK) cells are key effector cells in immune defenses against intracellular pathogens and cancer. In human blood, effector T and NK cytotoxic cells comprise a diverse and relatively rare group of cells. Herein, ... ...

    Abstract Cytotoxic T lymphocytes and natural killer (NK) cells are key effector cells in immune defenses against intracellular pathogens and cancer. In human blood, effector T and NK cytotoxic cells comprise a diverse and relatively rare group of cells. Herein, we describe a simplified intracellular staining workflow for classification of circulating human T and NK cells with cytolytic potential. We suggest reagents for measuring cytolytic proteins and identification of cell subsets within conventional and unconventional T cells and NK cells.
    Mesh-Begriff(e) Flow Cytometry ; Humans ; Killer Cells, Natural ; T-Lymphocytes, Cytotoxic
    Sprache Englisch
    Erscheinungsdatum 2021-12-29
    Erscheinungsland United States
    Dokumenttyp Journal Article
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2021.101069
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  7. Artikel ; Online: Co-staining human PBMCs with fluorescent antibodies and antibody-oligonucleotide conjugates for cell sorting prior to single-cell CITE-Seq.

    Shi, Xiaoshan / Baracho, Gisele V / Lomas, Woodrow E / Widmann, Stephanie J / Tyznik, Aaron J

    STAR protocols

    2021  Band 2, Heft 4, Seite(n) 100893

    Abstract: The dual interrogation of the transcriptome and proteome with single-cell resolution provides exquisite insights into immune mechanisms in health and disease. Here, we describe a cutting-edge protocol wherein we combine Cellular Indexing of ... ...

    Abstract The dual interrogation of the transcriptome and proteome with single-cell resolution provides exquisite insights into immune mechanisms in health and disease. Here, we describe a cutting-edge protocol wherein we combine Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), a technique utilizing antibody-oligonucleotide conjugates (AOCs), with fluorescence-activated cell sorting to enrich rare cell populations. Our protocol incorporates co-staining of cells with both fluorescent antibodies and AOCs simultaneously for subsequent input into the cell sorting and CITE-Seq pipeline. For complete details on the use and execution of this protocol, please refer to Mair et al. (2020).
    Mesh-Begriff(e) Epitopes/chemistry ; Flow Cytometry/methods ; Fluorescent Antibody Technique/methods ; Gene Expression Profiling/methods ; Humans ; Leukocytes, Mononuclear/chemistry ; Leukocytes, Mononuclear/cytology ; Single-Cell Analysis/methods
    Chemische Substanzen Epitopes
    Sprache Englisch
    Erscheinungsdatum 2021-10-19
    Erscheinungsland United States
    Dokumenttyp Journal Article
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2021.100893
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  8. Artikel ; Online: Functional phenotyping of circulating human cytotoxic T cells and NK cells using a 16-color flow cytometry panel

    Gisele V. Baracho / Nihan Kara / Stephanie Rigaud / Evelyn Lo / Stephanie J. Widmann / Aaron J. Tyznik

    STAR Protocols, Vol 3, Iss 1, Pp 101069- (2022)

    2022  

    Abstract: Summary: Cytotoxic T lymphocytes and natural killer (NK) cells are key effector cells in immune defenses against intracellular pathogens and cancer. In human blood, effector T and NK cytotoxic cells comprise a diverse and relatively rare group of cells. ... ...

    Abstract Summary: Cytotoxic T lymphocytes and natural killer (NK) cells are key effector cells in immune defenses against intracellular pathogens and cancer. In human blood, effector T and NK cytotoxic cells comprise a diverse and relatively rare group of cells. Herein, we describe a simplified intracellular staining workflow for classification of circulating human T and NK cells with cytolytic potential. We suggest reagents for measuring cytolytic proteins and identification of cell subsets within conventional and unconventional T cells and NK cells.
    Schlagwörter Single Cell ; Flow Cytometry/Mass Cytometry ; Cell-based Assays ; Cancer ; Health Sciences ; Immunology ; Science (General) ; Q1-390
    Sprache Englisch
    Erscheinungsdatum 2022-03-01T00:00:00Z
    Verlag Elsevier
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  9. Artikel ; Online: Co-staining human PBMCs with fluorescent antibodies and antibody-oligonucleotide conjugates for cell sorting prior to single-cell CITE-Seq

    Xiaoshan Shi / Gisele V. Baracho / Woodrow E. Lomas, III / Stephanie J. Widmann / Aaron J. Tyznik

    STAR Protocols, Vol 2, Iss 4, Pp 100893- (2021)

    2021  

    Abstract: Summary: The dual interrogation of the transcriptome and proteome with single-cell resolution provides exquisite insights into immune mechanisms in health and disease. Here, we describe a cutting-edge protocol wherein we combine Cellular Indexing of ... ...

    Abstract Summary: The dual interrogation of the transcriptome and proteome with single-cell resolution provides exquisite insights into immune mechanisms in health and disease. Here, we describe a cutting-edge protocol wherein we combine Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), a technique utilizing antibody-oligonucleotide conjugates (AOCs), with fluorescence-activated cell sorting to enrich rare cell populations. Our protocol incorporates co-staining of cells with both fluorescent antibodies and AOCs simultaneously for subsequent input into the cell sorting and CITE-Seq pipeline.For complete details on the use and execution of this protocol, please refer to Mair et al. (2020).
    Schlagwörter Cell isolation ; Single Cell ; Flow Cytometry/Mass Cytometry ; Immunology ; Molecular Biology ; Antibody ; Science (General) ; Q1-390
    Sprache Englisch
    Erscheinungsdatum 2021-12-01T00:00:00Z
    Verlag Elsevier
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  10. Artikel ; Online: AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level.

    Erickson, Jami R / Mair, Florian / Bugos, Grace / Martin, Jody / Tyznik, Aaron J / Nakamoto, Margaret / Mortimer, Stefanie / Prlic, Martin

    STAR protocols

    2020  Band 1, Heft 2

    Abstract: By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of ...

    Abstract By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 10
    Mesh-Begriff(e) Antibodies/immunology ; Gene Expression/genetics ; Gene Expression Profiling/methods ; High-Throughput Nucleotide Sequencing/methods ; Proteins/analysis ; Proteins/genetics ; Proteins/immunology ; Proteomics/methods ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/methods ; Transcriptome/genetics
    Chemische Substanzen Antibodies ; Proteins
    Sprache Englisch
    Erscheinungsdatum 2020-08-25
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2020.100092
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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