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

✍ Scribed by Mireille Verdier; Chantal Jayat; Marie-Helène Ratinaud; Danielle Troutaud

Publisher
John Wiley and Sons
Year
2000
Tongue
English
Weight
185 KB
Volume
41
Category
Article
ISSN
0196-4763

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✦ Synopsis

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.

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