Optimization of cell permeabilization for multiparametric flow cytometric analysis with lectin staining

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.