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T. A. Bogush, A. S. Shaturova, E. A. Dudko, E. E. Zhuraev, B. E. Polotsky, G. V. Ungiadze, M. I. Davydov

Quantitative immunofluorescence estimation of estrogen receptors β in human solid tumors by flow cytometry

Abstract

The adaptation of immunofluorescence analysis for estrogen receptors β (ERβ) quantitative estimation in biopsy specimens of human solid tumors by flow cytometry was performed. The primary monoclonal antibodies specific to the N-terminal domain of human ERβ protein (clone 14C8, Abcam) and the secondary FITC-conjugated antibodies (F2772, Sigma) were used in the investigation. We have shown the possibility of cell fixation in 4% formaldehyde solution, that enables long-term storage of tumor cell suspensions and, if necessary, verification of the results. It was shown also the possibility to decrease the cell concentration for the test (from 1´106 to 0,5´103 cells/ml) and the cell number for reliable detection of ERb by flow cytometer (from 1´104 to 1´103 cells). The later allows investigation both surgical and endoscopic biopsy specimens. It was found that the overnight cell incubation with the primary antibodies (1520 h), and with the secondary antibodies (1.5 h) optimizes the sensitivity of the method (as compared to 1.5 h incubation with each of the antibodies) and thereby let reduce the antibody consumption. That time-schedule of the incubation allows also revealing the all ERb expressing cells in the tumor sample investigated because of reaching the plateau region of the cell fluorescence and optimizing the investigation for the staff. The range of the primary antibody concentrations 1.2, 2.5 and 5.0 g/ml which covers both the linear and the plateau regions of the specific cell fluorescence in solid human tumor investigated is recommended for clinical testing. Human breast adenocarcinoma cell line MCF-7 and the range of the primary antibody concentrations 0.06, 1.2 and 2.5 g/ml is recommended for the regular antibody activity control in clinical using.
Moscow University Chemistry Bulletin.
2011, Vol. 52, No. 4, P. 305
   

Copyright (C) Chemistry Dept., Moscow State University, 2002
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