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A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry
G Halldén1, U Andersson, J Hed
1Department of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden.
Insights
This study introduces a novel cell membrane permeabilization method for detecting intracellular antigens using immunofluorescence and flow cytometry. The technique preserves cell integrity and antigenicity, enabling clear analysis of human leukocytes.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Intracellular antigen detection is crucial for understanding cell function and disease.
- Existing methods for cell membrane permeabilization can compromise cell integrity and antigenicity.
- Accurate analysis of leukocyte subpopulations requires methods that preserve cellular markers.
Purpose of the Study:
- To develop and validate a new, simple, and reproducible cell membrane permeabilization method.
- To enable reliable detection of intracellular antigens in human peripheral blood leukocytes.
- To preserve both intracellular antigenicity and cell surface marker expression.
Main Methods:
- Paraformaldehyde fixation to stabilize cell membranes and preserve antigenicity.
- Detergent treatment (n-octyl-beta-D-glucopyranoside) for controlled cell membrane permeabilization.
- Application to mixed leukocyte populations from human peripheral blood for immunofluorescence staining and flow cytometry.
Main Results:
- The method effectively permeabilizes the cell membrane without altering cell number or causing aggregation.
- Intracellular antigenicity is preserved, allowing detection of intracellular antigens like vimentin.
- Cell surface antigen expression remains unaltered, facilitating phenotypic characterization.
- Leukocyte subpopulations are well-separated and clearly detectable by flow cytometry.
Conclusions:
- This novel permeabilization technique offers a robust approach for intracellular antigen detection.
- The method is suitable for analyzing human peripheral blood leukocytes without prior purification.
- It enables accurate phenotypic characterization of cells based on both intracellular and cell surface markers.
Abstract:
This article describes a new cell membrane permeabilization method for the detection of intracellular antigens by immunofluorescence staining and flow cytometry. The number of cells remained unaltered and no cell aggregation or loss of intracellular antigenicity was observed after this permeabilization treatment. A mixed leukocyte population from human peripheral blood was used in this study and the leukocytes were fixed and permeabilized, which permitted monoclonal anti-vimentin antibodies to reach intracellular antigens. The stabilization of cell membranes and preservation of intracellular antigenicity was achieved with paraformaldehyde fixation. This pretreatment prevents cell destruction and subsequent treatment with the detergent n-octyl-beta-D-glucopyranoside results in permeabilization of the cell membrane. The procedure does not alter the expression of cell surface antigens, which is of importance if phenotypic characterization of intracellularly stained cells is to be undertaken. Furthermore, this simple, rapid and reproducible technique makes it possible to detect and distinguish between different human peripheral blood leukocytes without prior purification steps. The leukocyte subpopulations remain well-separated and easily detectable by flow cytometry.