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A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic
H Lecoeur1, E Ledru, M L Gougeon
1Unité d'Oncologie Virale, Département SIDA et Rétrovirus, Institut Pasteur, Paris, France.
Insights
This study presents a reliable method for detecting surface and intracellular antigens in apoptotic lymphocytes. Saponin permeabilization preserves cell morphology and allows accurate quantification and simultaneous analysis of multiple antigens.
Area of Science:
- Immunology
- Cell Biology
Background:
- Apoptosis in peripheral human lymphocytes is a critical process, particularly in conditions like HIV infection.
- Simultaneous detection of surface and intracellular antigens in apoptotic cells is challenging due to permeabilization procedures.
Purpose of the Study:
- To establish a simple and reliable method for simultaneous detection of surface and intracellular antigens in apoptotic human lymphocytes.
- To evaluate the impact of different permeabilization methods on lymphocyte apoptosis markers and antigen expression.
Main Methods:
- Comparison of three permeabilization methods (saponin, Triton X-100, ethanol) for their effects on apoptotic lymphocyte quantification and antigen detection.
- Analysis of surface antigens (CD3, CD4, CD8, Fas, CD45R0) and intracellular molecules (Bcl-2, IFNγ, TNFα, IL-2).
- Experiments conducted on peripheral blood mononuclear cells (PBMC) from HIV-infected donors.
Main Results:
- Saponin permeabilization uniquely preserved lymphocyte morphology (FSC/SSC) and allowed accurate apoptotic cell quantification (7-AAD staining).
- Saponin ensured reliable surface immunophenotyping with good antibody binding capacity.
- Saponin enabled proper detection of intracellular antigens (Bcl-2) and cytokines (IFNγ, TNFα, IL-2), and combined analysis of apoptotic nuclei, surface, and intracellular molecules.
Conclusions:
- Saponin permeabilization is the optimal method for simultaneous analysis of extracellular and intracellular antigens in apoptotic lymphocytes.
- Appropriate detergent selection and defined staining procedures are crucial for overcoming challenges in analyzing complex lymphoid populations.
Abstract:
The aim of this study was to define a simple and reliable method to detect simultaneously surface and intracellular antigens in apoptotic peripheral human lymphocytes. This approach requires a permeabilizing procedure for intracellular access of mAbs, which raises the important question of the influence of this procedure on parameters which identify apoptotic cells and on the surface expression of antigens. We compared the effects of three currently used permeabilizing methods (saponin quillaia bark 0.05%, Triton X-100 0.1, ethanol 70%) on the quantification of apoptotic lymphocytes, defined according to FSC/SSC criteria or following 7-AAD staining, and on the detection of surface CD3, CD4, CD8, Fas, CD45R0 molecules. The combined detection of these surface antigens with intracellular molecules, including Bcl-2 and cytokines (IFNgamma, TNFalpha, IL-2) was also analysed in the context of these three permeabilizing procedures. All the experiments were performed on PBMC from HIV-infected donors, known to undergo excessive apoptosis following short-term culture. We report that permeabilization with saponin is the only procedure which allows: (1) the preservation of lymphocyte morphology determined by the FSC/SSC parameters; (2) the quantification of apoptotic lymphocytes following 7-AAD staining; (3) a reliable surface immunophenotyping, maintaining a good antibody binding capacity (ABC); (4) the proper detection of intracellular membrane bound antigens (Bcl-2) and intracellular cytokines (IFNgamma, TNFalpha, IL-2); (5) the combined detection of apoptotic nuclei, surface antigens and intracellular molecules. Altogether these observations demonstrate that the simultaneous analysis of extracellular and intracellular antigens in apoptotic cells belonging to a complex lymphoid populations such as PBMC can be readily overcome provided the detergent used for cell permeabilization is appropriate and the successive staining procedures performed in a defined order.
