Related Experiment Video
Updated: Aug 8, 2026
Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry
Published on: June 8, 2012
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
Insights
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.
Background:
This study was undertaken in mice to develop a reproducible procedure of cell permeabilization, allowing intracellular protein staining by immunofluorescence (i.e., Bcl-2) without losing surface labeling especially for lectins (i.e., B220 and peanut agglutinin [PNA]). This article reports results obtained with different permeabilization protocols.
Methods:
Lymphoid cells were extracted and prepared from Peyer's patches and spleen. After surface labeling using anti-B220-Cy-chrome and PNA-biotin/streptavidin-phycoerythrin, we comparatively tested three permeabilization protocols: saponin 0.3%, methanol 70%, and the commercial kit Dako Intrastain. Final Bcl-2 staining was performed and cells were analyzed by flow cytometry.
Results:
With 0.3% saponin as the permeabilization reagent, a significant loss of lectin labeling was observed when comparing mono PNA and triple (i.e. , B220-PNA-Bcl-2) staining (74.8% and 22.5% positive cells, respectively). Quality of PNA staining was conserved with Intrastain when comparing multiparametric versus monoparametric stainings (82. 4% of positive cells versus 78.3%, respectively). Intrastain preserved scatter characteristics (69.9% of total cells in the lymphocyte gate with Intrastain versus 13.7% with saponin 0.3% and 20.9% with methanol 70%). This protocol has been used for a preliminary multiparametric analysis in order to quantify Bcl-2 expression in PNA/B220-positive cells.
Conclusion:
This protocol may be useful to assess simultaneously lectin cell surface labeling and intracellular target staining.
