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  1. Artikel ; Online: CODEX multiplexed tissue imaging with DNA-conjugated antibodies.

    Black, Sarah / Phillips, Darci / Hickey, John W / Kennedy-Darling, Julia / Venkataraaman, Vishal G / Samusik, Nikolay / Goltsev, Yury / Schürch, Christian M / Nolan, Garry P

    Nature protocols

    2021  Band 16, Heft 8, Seite(n) 3802–3835

    Abstract: Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and ... ...

    Abstract Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.
    Mesh-Begriff(e) Animals ; Antibodies/chemistry ; Biomarkers ; DNA/chemistry ; Diagnostic Imaging ; Haplorhini ; Humans ; Image Processing, Computer-Assisted ; Mice ; Paraffin Embedding ; Reproducibility of Results ; Single-Cell Analysis/methods ; Tissue Fixation/methods
    Chemische Substanzen Antibodies ; Biomarkers ; DNA (9007-49-2)
    Sprache Englisch
    Erscheinungsdatum 2021-07-02
    Erscheinungsland England
    Dokumenttyp Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, U.S. Gov't, P.H.S. ; Review
    ZDB-ID 2244966-8
    ISSN 1750-2799 ; 1754-2189
    ISSN (online) 1750-2799
    ISSN 1754-2189
    DOI 10.1038/s41596-021-00556-8
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  2. Artikel ; Online: Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction.

    Kennedy-Darling, Julia / Bhate, Salil S / Hickey, John W / Black, Sarah / Barlow, Graham L / Vazquez, Gustavo / Venkataraaman, Vishal G / Samusik, Nikolay / Goltsev, Yury / Schürch, Christian M / Nolan, Garry P

    European journal of immunology

    2021  Band 51, Heft 5, Seite(n) 1262–1277

    Abstract: Multiparameter tissue imaging enables analysis of cell-cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. ... ...

    Abstract Multiparameter tissue imaging enables analysis of cell-cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO-Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a LN. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single-cell analysis on the data, including unsupervised clustering, that revealed 31 cell types across all tissues. We compared cell-type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell-cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre-existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging.
    Mesh-Begriff(e) Antibodies ; Cell Communication ; Cell Count ; Histocytochemistry/methods ; Humans ; In Situ Hybridization/methods ; Lymphoid Tissue ; Molecular Imaging/methods ; Oligonucleotides ; Organ Specificity ; Reproducibility of Results ; Sensitivity and Specificity ; Single-Cell Analysis/methods
    Chemische Substanzen Antibodies ; Oligonucleotides
    Sprache Englisch
    Erscheinungsdatum 2021-03-10
    Erscheinungsland Germany
    Dokumenttyp Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, U.S. Gov't, P.H.S.
    ZDB-ID 120108-6
    ISSN 1521-4141 ; 0014-2980
    ISSN (online) 1521-4141
    ISSN 0014-2980
    DOI 10.1002/eji.202048891
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  3. Artikel ; Online: Organization of the human intestine at single-cell resolution.

    Hickey, John W / Becker, Winston R / Nevins, Stephanie A / Horning, Aaron / Perez, Almudena Espin / Zhu, Chenchen / Zhu, Bokai / Wei, Bei / Chiu, Roxanne / Chen, Derek C / Cotter, Daniel L / Esplin, Edward D / Weimer, Annika K / Caraccio, Chiara / Venkataraaman, Vishal / Schürch, Christian M / Black, Sarah / Brbić, Maria / Cao, Kaidi /
    Chen, Shuxiao / Zhang, Weiruo / Monte, Emma / Zhang, Nancy R / Ma, Zongming / Leskovec, Jure / Zhang, Zhengyan / Lin, Shin / Longacre, Teri / Plevritis, Sylvia K / Lin, Yiing / Nolan, Garry P / Greenleaf, William J / Snyder, Michael

    Nature

    2023  Band 619, Heft 7970, Seite(n) 572–584

    Abstract: The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall ... ...

    Abstract The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health
    Mesh-Begriff(e) Humans ; Cell Differentiation/genetics ; Chromatin/genetics ; Epithelial Cells/cytology ; Epithelial Cells/metabolism ; Gene Expression Regulation ; Intestinal Mucosa/cytology ; Intestines/cytology ; Intestines/immunology ; Single-Cell Analysis ; Single-Cell Gene Expression Analysis
    Chemische Substanzen Chromatin
    Sprache Englisch
    Erscheinungsdatum 2023-07-19
    Erscheinungsland England
    Dokumenttyp Journal Article
    ZDB-ID 120714-3
    ISSN 1476-4687 ; 0028-0836
    ISSN (online) 1476-4687
    ISSN 0028-0836
    DOI 10.1038/s41586-023-05915-x
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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