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

ABSTRACT: 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 fu

SUBMITTER: Folkerts J 

PROVIDER: S-EPMC7197894 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature