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A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes
- Source :
- Journal of immunological methods. 217(1-2)
- Publication Year :
- 1998
-
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 (IFN γ , TNF α , 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 (IFN γ , TNF α , 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.
- Subjects :
- Intracellular Fluid
Cell Membrane Permeability
Octoxynol
CD3
Lymphocyte
Immunology
Detergents
Apoptosis
HIV Infections
Biology
Lymphocyte Activation
Peripheral blood mononuclear cell
Flow cytometry
Immunophenotyping
Antigen
Antigens, CD
medicine
Extracellular
Immunology and Allergy
Humans
Lymphocytes
fas Receptor
Antigens
Fluorescent Antibody Technique, Indirect
Fluorescent Dyes
Lymphokines
medicine.diagnostic_test
Ethanol
Antibodies, Monoclonal
Saponins
Flow Cytometry
Molecular biology
medicine.anatomical_structure
Proto-Oncogene Proteins c-bcl-2
Antigens, Surface
biology.protein
Dactinomycin
Intracellular
Subjects
Details
- ISSN :
- 00221759
- Volume :
- 217
- Issue :
- 1-2
- Database :
- OpenAIRE
- Journal :
- Journal of immunological methods
- Accession number :
- edsair.doi.dedup.....a4a7ee332a7e96f1c7fbccea6519e57f