Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells

✍ Scribed by Elizabeth C. de Boer; Rob F. M. Bevers; Karl-Heinz Kurth; Denis H. J. Schamhart

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 645 KB
• Volume: 25
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19961201)25:4<381::aid-cyto10>3.0.co;2-r

✦ Synopsis

This study describes a new flow cytometric method for assessment of phagocytosis of specific bacteria (bacillus Calmette-Gukrin (BCG) and Eschericbiu coZi) by bladder epithelial cells. The internalization assay consisted of labeling bacteria chemically with fluorescein hthiocyanate (FITC). Subsequent to incubation of fluoresceinated bacteria with internalizing cells, adherent nonphagocytosed bacteria were marked by two-step labeling using specific antibodies and phycoerythrin (PE)conjugated antibodies. Double fluorescent FACS analysis differentiated between bacterial phagocytosis and adherence. The validity of the method was shown by inhibition of BCG phagocytosis at 4°C by cytochalasin B, by removal of excess free bacteria, and by anti-BCG antibodies. BCG-phagocytizing and -nonphagocytizing cell lines were discrimhated by applying this technique to a series of bladder carcinoma cell lines. There seemed to be a relationship between phagocytic capacity and grade of differentiation in these cell lines, which may have implications for topical BCG immunotherapy in superficial bladder cancer. In conclusion, a new, reliable, rapid, and relatively simple double fluorescent method is described for quanti5cation of specific bacterial internalization by large numbers of (bladder) epithelial cells. This method should be generally applicable to the study of in vitro interaction between bacteria and Merent types of host cells.