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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
Simultaneous measurement of cell surface and intracellular antigens by multiple flow cytometry
H Sumner1, D Abraham, G Bou-Gharios
1Cell Enzymology Unit, Kennedy Institute of Rheumatology, London, U.K.
Insights
This study introduces a saponin-based method for simultaneous detection of intracellular and surface antigens in lymphocytes using flow cytometry (FCM). Activated T cells showed increased expression of CD2 and beta-glucuronidase (Gus).
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Flow cytometry (FCM) is a powerful technique for analyzing cell populations.
- Simultaneous detection of intracellular and surface antigens is crucial for understanding cellular processes.
- Existing methods may have limitations in sensitivity or specificity for multi-antigen detection.
Purpose of the Study:
- To develop and validate a novel method for permeabilizing lymphocytes using saponin.
- To enable simultaneous detection of intracellular proteins and surface antigens by FCM.
- To investigate the expression of CD2 and beta-glucuronidase (Gus) during T cell activation.
Main Methods:
- Lymphocyte permeabilization using the detergent saponin.
- Flow cytometry (FCM) with monoclonal antibodies against CD2 and beta-glucuronidase (Gus).
- Immunogold electron microscopy for ultrastructural localization and validation.
- Three-color FCM for analyzing T cell subsets (CD4 and CD8).
Main Results:
- The saponin-based method allows accurate, simultaneous detection of surface (CD2) and intracellular (Gus) antigens.
- T cell activation leads to a marked increase in both CD2 and Gus expression.
- Immunogold electron microscopy confirmed the specificity and accuracy of the multi-labeling FCM procedure.
- Changes in Gus expression were observed in CD4 and CD8 T cell subsets upon activation.
Conclusions:
- Saponin-based FCM is a reliable method for simultaneous surface and intracellular antigen detection in lymphocytes.
- This technique provides insights into T cell activation dynamics and antigen expression.
- The findings contribute to a better understanding of immune cell responses and antigen localization.
Abstract:
We have employed a method for permeabilizing lymphocytes with the detergent saponin in order to detect an intracellular protein simultaneously with surface antigens by flow cytometry (FCM). Using monoclonal antibodies specific for the murine CD2 receptor and for the lysosomal enzyme, beta-glucuronidase (Gus), we found that the expression of both of these antigens increased markedly when T cells were activated. Two sensitive methods were used to show that FCM provided an accurate measure of the actual number of CD2 and Gus molecules present in the lymphocytes. Immunogold electron microscopy revealed the precise ultrastructural localization of these different components and corroborated the specificity of the multiple labelling procedure for the simultaneous detection of surface and intracellular antigens. We also developed a three-colour FCM technique which we used to examine the changes in Gus expression in the CD4 and CD8 T cell sub-sets during activation.
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