期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 94, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/52241
关键词
Neuroscience; Issue 94; CD markers; surface antigens; intracellular antigens; flow cytometry; neurons; glial cells; neural stem cells; fluorescence-activated cell sorting (FACS); CD24; CD54; CFSE
资金
- Emmy Noether-Program of the German Research Foundation (DFG) [PR1132/3-1]
- Muller-Fahnenberg Foundation of the University of Freiburg
- Excellence Initiative of the German Research Foundation (Spemann Graduate School) [GSC-4]
Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.
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