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. Conclusion This protocol may be useful to assess simultaneously lectin cell surface labeling and intracellular target staining. Cytometry 41:55–61, 2000 © 2000 Wiley‐Liss, Inc.

Retinoschisin, a Photoreceptor-Secreted Protein, and Its Interaction with Bipolar and Müller Cells

“…For these reasons we examined the presence of retinoschisin inside and outside photoreceptor, bipolar, and Müller cells by immunostaining dissociated cells that had or had not been permeabilized with methanol. This treatment has been shown to remove some proteins local-ized to the cell surface [i.e., the lectin peanut agglutinin (Verdier et al, 2000)] and to allow the detection of intracellular proteins Schipper et al, 1999). Consistent with these observations, we found that immunofluorescent staining of the cell-surface protein ROB was substantially reduced by methanol permeabilization (data not shown) and that intracellular CRALBP could be detected after methanol treatment.…”

Immunomagnetic enrichment and flow cytometric characterization of mouse microglia

“…We avoided the use of intracellular staining here because permeabilization conditions have been shown to critically affect results in both B cells (Verdier et al, 2000) and T cells (Papagno et al, 2007), suggesting that optimization of intracellular flow cytometry protocols for microglia/macrophages will not be trivial. Additional benefits of limiting the protocol to surface stains are the retained ability to sort live cells for functional experiments, as well as a significant reduction in time and labor.…”

Aged mice exhibit distinct peripheral B‐cell phenotypes differing in apoptotic susceptibility: An ex vivo analysis

“…Evaluation of Bcl-2 content was assessed on B-cells, after a fixation/permeabilization step that we performed with a commercial kit (Intrastain, Dako, Trappes, France) previously tested in our laboratory (20). Permeabilization was undertaken simultaneously with hamster anti-mouse Bcl-2-uncoupled monoclonal antibody (Pharmingen, 1 lg/10 6 cells) for 15 min at room temperature in the dark.…”