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Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types
Published on: December 18, 2014
Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types
Vishal Menon1, Ria Thomas2, Arun R Ghale3
1Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg.
Insights
This study details flow cytometry protocols for analyzing neural cell antigens. It provides a guide for neurobiologists to effectively use this technique for cell population analysis.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Flow cytometry is a standard technique for cell population analysis in various fields.
- Specific protocols for neural cell types are less common, hindering broader application in neuroscience.
Purpose of the Study:
- To provide detailed, step-by-step flow cytometry protocols for analyzing neural cell surface and intracellular antigens.
- To enable neurobiologists with limited flow cytometry experience to utilize this methodology effectively.
Main Methods:
- Harvesting of neural in vitro cultures.
- Optional carboxyfluorescein succinimidyl ester (CFSE)-labeling.
- Surface antigen staining using conjugated cluster of differentiation (CD) antibodies (e.g., CD24, CD54).
- Intracellular antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling).
- Explanation of experimental planning, controls, gating strategies, and compensation.
Main Results:
- A comprehensive protocol for flow cytometric analysis of neural cell antigens is presented.
- The methodology covers both surface and intracellular antigen detection.
- Key principles for experimental design and data analysis are explained.
Conclusions:
- This protocol empowers neurobiologists to confidently apply flow cytometry for neural cell analysis.
- The detailed guide and accompanying video facilitate the adoption of advanced flow cytometry techniques in neuroscience research.
Abstract:
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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