Publications

Publications, Books, Book Chapters and Reviews by Prof. Marcus Maurer, MD

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Rapid identification of human mast cell degranulation regulators using functional genomics coupled to high-resolution confocal microscopy

Filename 380. Folkerts et al., Rapid ident. human MC confocal micr., FrontProt2020.pdf
Filesize 2 MB
Version o.380
Date added June 4, 2019
Downloaded 2 times
Category Original Work
Tags IgE-dependent, mast cell
Authors Folkerts, J., Gaudenzio, N., Maurer, M., Hendriks, R. W., Stadhouders, R. Tam, S. Y., and Galli, S. J.
Citation Folkerts, J., Gaudenzio, N., Maurer, M., Hendriks, R. W., Stadhouders, R. Tam, S. Y., and Galli, S. J.: Rapid identification of human mast cell degranulation regulators using functional genomics coupled to high-resolution confocal microscopy. Nat. Protoc. 2020: 15; 1285-1310.
Corresponding authors Galli, S. J.
DocNum o.380
DocType PDF
Edition; Page 15; 1285-1310
IF 13.49
Publisher Nat. Protoc.
ReleaseDate 2020

Targeted functional genomics represents a powerful approach for studying gene function in vivo and in vitro. However, its application to gene expression studies in human mast cells has been hampered by low yields of human mast cell cultures and their poor transfection efficiency. We developed an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR–Cas9 gene editing. By using high-resolution confocal microscopy and a fluorochrome-labeled avidin probe, one can directly assess the alteration of functional responses, i.e., degranulation, in single human mast cells (10–12 weeks old). The elimination of a drug or marker selection step avoids the use of potentially toxic treatment procedures, and the brief hands-on time of the functional analysis step enables highthroughput screening of shRNA or CRISPR–Cas9 constructs to identify genes that regulate human mast cell degranulation. The ability to analyze single cells substantially reduces the total number of cells required and enables the parallel visualization of the degranulation profiles of both edited and non-edited mast cells, offering a consistent internal control not found in other protocols. Moreover, our protocol offers a flexible choice between RNA interference (RNAi) and CRISPR–Cas9 genome editing for perturbation of gene expression using our human mast cell single-cell imaging system. Perturbation of gene expression, acquisition of microscopy data and image analysis can be completed within 5 d, requiring only standard laboratory equipment and expertise.

(Last update: 08.2021)

Number of publications (original work and reviews) in peer-reviewed journals: 636
Number of original publications in peer-reviewed journals: 462
Number of reviews in peer-reviewed journals: 174
Cumulative IF of publications (original work & reviews) in peer-reviewed journals: 3834,12
Cumulative IF for original publications in peer-reviewed journals: 3043,14
Cumulative IF for reviews in peer-reviewed journals: 790,98
Citations, Hirsch index: (view on Web of Science) 26429