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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
(15–20 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 µg/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 µg/ml is recommended for the regular antibody
activity control in clinical using.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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