Detection of bromodeoxyuridine-labeled cells by differential fluorescence analysis of DNA fluorochromes.

摘要: Publisher Summary This chapter describes a sensitive two-color flow cytometry (FCM) method for detecting Bromodeoxyuridine (BrdUrd)-labeled DNA in cells treated ≤ 30 minutes. The technique uses two nonintercalating, specific fluorochromes: Hoechst 33342 (HO) and gas chromatography (GC)-binding mithramycin (MI), dye whose fluorescence the presence of BrdUrd remains stoichiometric to content, under conditions employed. Using dual-wavelength excitation, blue HO green–yellow MI emissions are measured, differential amplifier subtracts from signal amplitude on cell-by-cell basis; resulting difference is amplified. Cells S-phase exhibit significant BrdUrd-HO quenching produce greater compared with that G 1 2 + M phase, which, except minor differences stainability, show relatively small differences. simple rapid requires only one-step staining. It mild therefore minimizes cell loss other important cellular markers, such as and/or chromatin, RNA, proteins, including antigens.