A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells

Journal Article · Sun Nov 01 04:00:00 EST 1987 · Experimental Cell Research; (United States)

DOI:https://doi.org/10.1016/0014-4827(87)90350-8· OSTI ID:5745242

Crissman, H A; Steinkamp, J A ^[1]

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1. Los Alamos National Lab., NM (United States)

A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G{sub 1} and G{sub 2}{plus}M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.

• OSTI ID: 5745242
• Journal Information: Experimental Cell Research; (United States), Journal Name: Experimental Cell Research; (United States) Vol. 173:1; ISSN ECREA; ISSN 0014-4827
• Country of Publication: United States
• Language: English

Related Subjects

550301* -- Cytology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANTIMETABOLITES
AUTORADIOGRAPHY
AZINES
BROMOURACILS
BUDR
CELL CYCLE
CELL FLOW SYSTEMS
CHO CELLS
CHROMATIN
DNA REPLICATION
DRUGS
FLUORESCENCE
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
LUMINESCENCE
NUCLEIC ACID REPLICATION
NUCLEOSIDES
NUCLEOTIDES
ORGANIC BROMINE COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PYRIMIDINES
RIBOSIDES
UPTAKE
URACILS