Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
Ultraviolet-induced detection of halogenated pyrimidines: Simultaneous analysis of DNA replication and cellular markers
Autore:
Hammers, HJ; Kirchner, H; Schlenke, P;
Indirizzi:
Med Univ Lubeck, Sch Med, Inst Immunol & Transfus Med, D-23538 Lubeck, Germany Med Univ Lubeck Lubeck Germany D-23538 sfus Med, D-23538 Lubeck, Germany
Titolo Testata:
CYTOMETRY
fascicolo: 4, volume: 40, anno: 2000,
pagine: 327 - 335
SICI:
0196-4763(20000801)40:4<327:UDOHPS>2.0.ZU;2-E
Fonte:
ISI
Lingua:
ENG
Soggetto:
SUBSTITUTED SV40 DNA; SINGLE-STRANDED-DNA; DIAGNOSTIC-TOOL; INCORPORATED BROMODEOXYURIDINE; BRDURD INCORPORATION; EXONUCLEASE-III; CYCLE ANALYSIS; CELLS; MEDICINE; ANTIBODY;
Keywords:
BrdUrd; ultraviolet; cell cycle; flow cytometry; immunophenotyping;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
36
Recensione:
Indirizzi per estratti:
Indirizzo: Hammers, HJ Med Univ Lubeck, Sch Med, Inst Immunol & Transfus Med, Ratzeburger Allee 160, D-23538 Lubeck, Germany Med Univ Lubeck Ratzeburger Allee 160 Lubeck Germany D-23538
Citazione:
H.J. Hammers et al., "Ultraviolet-induced detection of halogenated pyrimidines: Simultaneous analysis of DNA replication and cellular markers", CYTOMETRY, 40(4), 2000, pp. 327-335

Abstract

Background: We describe a new nonenzymatic method ology that allows the simultaneous detection of DNA replication and other cellular markers such as immunophenotyping. DNA replicating cells are identified by their incorporation of halogenated thymidine analogs, e.g., 5-bromo-deoxyuridine (BrdUrd). Methods: Irradiation with ultraviolet (UV)-B or UV-A light in the presenceof Hoechst 33258 and subsequent treatment with a hypotonic buffer makes BrdUrd accessible to monoclonal antibodies (mAb), thus allowing its sensitivedetection. Results: The photolysis of BrdUrd in DNA with UV light is sequence dependent and results in DNA damage, allowing the detection of remaining BrdUrd using hypotonic conditions. However, treatment with other inducers of single or double- strand breaks of DNA such as gamma irradiation or hydrogen peroxide did not allow BrdUrd detection. The new methodology is compatible with both mild crosslinking fixation, i.e., aldehydes, or coagulative fixation, i.e., alcohols. The successful identification of CD34+, CD138+, or CD19+ cells out of heterogeneous cell suspensions and their cell-cycle analysis aredescribed. Results correlated very well with acid denaturation (r = 0.972). The average coefficient of variation (CV) of G(1) in the DNA histogram was smaller than 5%, resulting in good preservation of DNA distribution. Also, the signal-to-noise ratio was almost twice as high as for 2N acid denaturation, facilitating convenient discrimination of BrdUrd-positive cells. Conclusions: In contrast to previous approaches, this methodology eliminates the need for anp additional enzymatic treatment such as DNA digestion orstrand-break labeling after UV irradiation. The method is fast, convenient, and inexpensive and should be able to promote the use of halogenated pyrimidines in basic and clinical research of cancer, immunology, and pharmacology. (C) 2000 Wiley Liss, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 15/08/20 alle ore 19:55:57