期刊
NATURE PROTOCOLS
卷 12, 期 6, 页码 1245-1260出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.039
关键词
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资金
- National Institutes of Health [AI-104615, AI-106036, AI-124691]
- Rutgers Office of Research and Economic Development/New Jersey Health Foundation
We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes similar to 30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.
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