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Article ; 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.
Language	English
Publishing date	2024-04-18
Publishing country	United States
Document type	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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Article ; 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 Volume 2779, Page(s) 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 term(s)	Gene Expression Profiling/methods ; Epitopes ; Transcriptome ; Cell Separation ; Antibodies ; Single-Cell Analysis/methods
Chemical Substances	Epitopes ; Antibodies
Language	English
Publishing date	2024-04-01
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3738-8_13
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Article: 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 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.
Language	English
Publishing date	2023-12-15
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.12.14.571745
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Article ; Online: High-Dimensional Immunophenotyping with Fluorescence-Based Cytometry: A Practical Guidebook.

Mair, Florian / Tyznik, Aaron J

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

2019 Volume 2032, Page(s) 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 term(s)	Flow Cytometry/methods ; Fluorescence ; Immunophenotyping/methods ; Molecular Biology/methods ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/methods
Language	English
Publishing date	2019-08-08
Publishing country	United States
Document type	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
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; 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 Volume 105, Issue 1, Page(s) 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 term(s)	Flow Cytometry/methods ; Epitopes ; Antibodies ; Cell Separation/methods ; T-Lymphocytes ; Antigens ; Single-Cell Analysis
Chemical Substances	Epitopes ; Antibodies ; Antigens
Language	English
Publishing date	2023-09-29
Publishing country	United States
Document type	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
Database	MEDical Literature Analysis and Retrieval System OnLINE

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In stock of ZB MED Cologne/Königswinter

Zs.A 1688: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; 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 Volume 3, Issue 1, Page(s) 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 term(s)	Flow Cytometry ; Humans ; Killer Cells, Natural ; T-Lymphocytes, Cytotoxic
Language	English
Publishing date	2021-12-29
Publishing country	United States
Document type	Journal Article
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2021.101069
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Article ; 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 Volume 2, Issue 4, Page(s) 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 term(s)	Epitopes/chemistry ; Flow Cytometry/methods ; Fluorescent Antibody Technique/methods ; Gene Expression Profiling/methods ; Humans ; Leukocytes, Mononuclear/chemistry ; Leukocytes, Mononuclear/cytology ; Single-Cell Analysis/methods
Chemical Substances	Epitopes
Language	English
Publishing date	2021-10-19
Publishing country	United States
Document type	Journal Article
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2021.100893
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; 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.
Keywords	Single Cell ; Flow Cytometry/Mass Cytometry ; Cell-based Assays ; Cancer ; Health Sciences ; Immunology ; Science (General) ; Q1-390
Language	English
Publishing date	2022-03-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; 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).
Keywords	Cell isolation ; Single Cell ; Flow Cytometry/Mass Cytometry ; Immunology ; Molecular Biology ; Antibody ; Science (General) ; Q1-390
Language	English
Publishing date	2021-12-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; 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 Volume 1, Issue 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 term(s)	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
Chemical Substances	Antibodies ; Proteins
Language	English
Publishing date	2020-08-25
Publishing country	United States
Document type	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
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