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Updated: Aug 8, 2026
Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types
Published on: December 18, 2014
A flow cytometric technique for simultaneous analysis of human mononuclear cell surface antigens and DNA
K M Rigg1, B K Shenton, I A Murray
1Department of Surgery, Medical School, Newcastle upon Tyne, U.K.
Insights
This study introduces a simple, rapid method for dual staining of mononuclear cells, enabling cell cycle analysis of lymphocyte subpopulations. The technique uses fluorescein-conjugated antibodies and propidium iodide with saponin for reproducible results.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Flow cytometry is crucial for analyzing lymphocyte populations.
- Assessing cell cycle status alongside phenotypic markers is valuable for research.
- Existing methods may be time-consuming or compromise antigen integrity.
Purpose of the Study:
- To develop a straightforward and efficient dual-staining technique.
- To enable simultaneous analysis of lymphocyte surface markers and DNA content.
- To facilitate cell cycle analysis within specific lymphocyte subpopulations.
Main Methods:
- Mononuclear cells were stained with fluorescein-conjugated monoclonal antibodies.
- Saponin was used to permeabilize cells for propidium iodide uptake.
- Propidium iodide was employed to stain cellular DNA.
- Flow cytometry was utilized for data acquisition and analysis.
Main Results:
- The described method allows for dual staining of lymphocytes for phenotypic markers and DNA.
- Saponin effectively permeabilizes cells within 1 minute, preserving surface antigen expression.
- The technique is effective across a wide range of saponin concentrations (0.001%–1%).
- Cell cycle analysis of individual lymphocyte subpopulations was successfully achieved.
Conclusions:
- This novel dual-staining method is simple, rapid, and yields reproducible results.
- The technique is suitable for analyzing lymphocyte phenotypes and cell cycle status concurrently.
- It offers a valuable tool for immunological and cell biology research.
Abstract:
A method for dual staining of mononuclear cells for lymphocyte phenotypic markers and DNA is described. The cells were stained with fluorescein-conjugated monoclonal antibodies and then rendered permeable to propidium iodide using saponin. Propidium iodide stains DNA and, using flow cytometry, cell cycle analysis of individual lymphocyte subpopulations can be determined. Saponin acts within 1 min, preserves expression of surface antigens and is effective at all concentrations from 0.001% to 1%. This technique is simple, rapid and gives reproducible results.
