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

Background: This study was undertaken in mice to develop a reproducible pro cedure of cell permeabilization, allowing intracellular protein staining by immunofluorescence (i.e., Bcl-2) without losing surface labeling especiall y for lectins (i.e., B220 and peanut agglutinin [PNA]). This article report s results obtained with different permeabilization protocols. Methods: Lymphoid cells were extracted and prepared from Peyer's patches an d spleen. After surface labeling using anti-B220-Cy-chrome and PNA-biotin/s treptavidinphycoerythrin, we comparatively tested three permeabilization pr otocols: saponin 0.3%, methanol 70%, and the commercial kit Dako Intrastain . Final Bcl-2 staining was performed and cells were analyzed by flow cytome try. Results: With 0.3% saponin as the permeabilization reagent, a significant l oss 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 multi parametric versus monoparametric stainings (82.4% of positive cells versus 78.3%, respective ly). Intrastain preserved scatter characteristics (69.9% of total cells in the lymphocyte gate with Intrastain versus 13.7% with sap onin 0.3% and 20.9% with methanol 70%). This protocol has been used for a p reliminary multiparametric analysis in order to quantify Bcl-2 expression i n PNA/B220-positive cells. Conclusion: This protocol may be useful to assess simultaneously lectin cel l surface labeling and intracellular target staining. (C) 2000 Wiley-Liss, Inc.