Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Cytometry
M NüsseK Ruoss

Abstract

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified B...Continue Reading

References

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Citations

Oct 1, 1996·Cell Proliferation·W GorczycaZ Darzynkiewicz
Jul 23, 2013·Journal of Clinical Biochemistry and Nutrition·Kyoko YamashitaShinya Toyokuni
Jun 22, 2010·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·R M ZuckerW K Boyes
Apr 24, 2014·Analytical Biochemistry·Kyle D DukesGeorge Chumanov
Jun 2, 2018·International Journal for Numerical Methods in Biomedical Engineering·Fabio ManginiFabrizio Frezza

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