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Optimization of cell permeabilization for multiparametric flow cytometric analysis with lectin staining
M Verdier1, C Jayat, M H Ratinaud
1EP CNRS 118, Faculté des Sciences, Limoges, France. mireille.verdier@unilim.fr
Cytometry
|August 16, 2000
Summary
A new cell permeabilization protocol using Dako Intrastain effectively stains intracellular Bcl-2 while preserving surface lectin markers like B220 and peanut agglutinin (PNA) in mice lymphoid cells.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Developing reproducible cell permeabilization methods is crucial for multi-target immunofluorescence staining.
- Simultaneous intracellular and surface antigen detection is challenging, particularly with lectin surface markers.
Purpose of the Study:
- To establish a reliable cell permeabilization protocol for mice lymphoid cells.
- To enable simultaneous intracellular protein (Bcl-2) and surface lectin (B220, PNA) staining without compromising signal integrity.
Main Methods:
- Lymphoid cells from Peyer's patches and spleen were surface labeled with anti-B220 and peanut agglutinin (PNA).
- Three permeabilization methods (saponin, methanol, Dako Intrastain) were compared for their ability to retain surface and intracellular staining.
- Flow cytometry was used to analyze cells after staining for B220, PNA, and intracellular Bcl-2.
Main Results:
- Saponin permeabilization led to significant loss of PNA lectin labeling.
- Dako Intrastain effectively preserved PNA lectin staining and cell scatter characteristics compared to saponin and methanol.
- Intrastain allowed for multiparametric analysis, quantifying Bcl-2 expression in B220/PNA-positive cells.
Conclusions:
- The Dako Intrastain protocol is suitable for simultaneous surface lectin and intracellular target immunofluorescence staining.
- This method facilitates robust multiparametric analysis of lymphoid cell populations.