title: Optimization of cell permeabilization for multiparametric flow cytometric analysis with lectin staining.
abstract: 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.
  
  
  CONCLUSIONS
  This protocol may be useful to assess simultaneously lectin cell surface labeling and intracellular target staining.
authors.affiliation: EP CNRS 118, Faculté des Sciences, Limoges, France. mireille.verdier@unilim.fr
authors.forename: M
authors.initials: M
authors.lastname: Verdier
authors.forename: C
authors.initials: C
authors.lastname: Jayat
authors.forename: M H
authors.initials: MH
authors.lastname: Ratinaud
authors.forename: D
authors.initials: D
authors.lastname: Troutaud
mesh_terms: D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects; D003593:Cytoplasm / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards*; D005455:Fluorescent Antibody Technique; D018858:Germinal Center / Q000166:cytology; D008565:Membrane Proteins / Q000032:analysis; D000432:Methanol; D051379:Mice; D008807:Mice, Inbred BALB C; D019887:Peanut Agglutinin; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis; D012503:Saponins; D012997:Solvents; D013154:Spleen / Q000166:cytology; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D019887:Peanut Agglutinin; D019253:Proto-Oncogene Proteins c-bcl-2; D012503:Saponins; D012997:Solvents; D019009:Proto-Oncogene Proteins c-kit; D000432:Methanol