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.

Chronology of the apoptotic events induced in the K562 cell line by photodynamic treatment with hematoporphyrin and monoglucosylporphyrin.

Photodynamic treatment of promyelocytic K562 cells in the presence of a monoglucosylporphyrin or hematoporphyrin leads to a sequence of events recognized as hallmarks of apoptosis: a drop in mitochondrial potential, concurrent with a drop in ATP level and a decrease in cell respiration, translocation of phosphatidylserine of the plasma membrane, DNA fragmentation, appearance of apoptotic bodies and eventually loss of plasma membrane integrity. The chronology of these events is in accordance with sequential events induced by other known proapoptotic agents; in contrast to these agents that induce apoptosis in a restricted part of the cell population, we observed that the entire cell population (apart from a small percentage of cells that endured rapid necrosis during phototreatment) underwent apoptosis after irradiation in the presence of porphyrins. It appears that photodynamic treatment allows the bypass of early apoptotic signals in K562 cells that are otherwise renowned for their resistance to drug-induced apoptosis (A. McGahon, R. Bissonnette, M. Schmitt, K. M. Cotter, D. R. Green and T. G. Cotter, Blood 83, 1179-1187, 1994). Singlet oxygen is believed to be the proximate reactive species generated by porphyrin illumination. Because this molecule reacts with almost every cellular constituent, the way that singlet oxygen or its reactive oxygen species byproducts trigger apoptosis remains to be elucidated.

Cell cycle analysis by flow cytometry: principles and applications.

Numerous flow cytometric analyses are based on DNA content studies. We have considered firstly monoparametric cell cycle analyses, which only take DNA content into account, but are sometimes of limited interest. Then, we have presented multiparametric analyses, which can be used to improve cycle phase identification by taking simultaneously into account DNA and other cellular components, or by considering some events occurring during cell cycle. Finally, we have discussed monoparametric and multiparametric cell cycle analysis interest in various application fields, particularly in pharmacology, toxicology, tumoral pathology and higher plant system studies.

Recent advances of flow cytometry in fundamental and applied microbiology.

This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated: light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology: fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

On-line visualization of the competitive behavior of antagonistic bacteria.

To study the interaction between cocultured Listeria monocytogenes and an antagonistic Leuconostoc strain producing an anti-Listeria bacteriocin, flow cytometry, a technique allowing on-line and real-time analysis, was used along with classical microbiological methods. Culture methods and flow cytometric measurements of the mixed culture over time point to a bactericidal action of the lactic acid-producing bacterial strain against L. monocytogenes cells.