Rapid fluorometric detection of drug resistant tumour cells

deFazio, Anna; Tattersall, Martin H. N.

doi:10.1038/bjc.1985.238

Cited by 11 publications

(1 citation statement)

References 18 publications

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“…DNases were applied to the cells in the following reaction solutions: 100 mM NaC1, 300 mM sucrose, 3 mM M g 4 + (pH 7.5), for DNase I; 60 mM Tris-HC1, 6 mM Mg4 + (pH 8.0), for exonuclease 111; 50 mM sodium acetate, 28 mM NaC1, 1 mM Z n t + , 0.5% glycerol (pH 5.7), for S1 nuclease; and 50 mM Tris-HC1, 50 mM NaC1, 10 mM Mg++ (pH 8.01, for Pstl. The reactions were carried out for 1 h at 23°C for DNase I or at 37°C for other enzymes and stopped by washing thoroughly in PBS containing 20 mM EDTA at 4°C.…”

Section: Dnase Treatment Of Cellsmentioning

confidence: 99%

Detection of 5‐bromo‐2‐deoxyuridine (BrdUrd) incorporation with monoclonal anti‐brdurd antibody after deoxyribonuclease treatment

et al. 1993

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We studied the effects of deoxyribonucleases on the detection of 5-bromo-2-deoxyuridine (BrdUrd) by anti-BrdUrd monoclonal antibodies (mAbs). After DNase I treatment, BrdUrd was detected in cells fixed on slides with the antiBrdUrd mAbs, B44 and BMC9318. The level of detection related to the degree of DNA digestion. DNA digestion of 25-75% resulted in levels of staining comparable to control preparations in which DNA was denatured by heating with formamide. Staining with the mAbs of DNase I-treated cells was abolished with S1 nuclease, a single-stranded DNA-specific nuclease. When exonuclease I11 was used after DNase I treatment, the staining intensity of cells fixed on slides increased, and BrdUrd could be detected in suspended cells by flow cytometry. Since this enzymatic method leading to the detection of BrdUrd does not involve cell loss, or destruction of either cellular morphology or epitope reactivity, as occurs with traditional DNA denaturation procedures, it is useful for kinetic studies of phenotypically mixed populations. Furthermore, staining with anti-BrdUrd mAb of cells treated with exonuclease I11 offers a simple approach to quantitation of apoptotic cells, in which an endogenous endonuclease is activated. Key terms: Flow cytometry, two-color analysis, exonuclease 111, single-stranded DNA, apoptosis 5-Bromo-2-deoxyuridine (BrdUrd), a thymidine analogue, which is incorporated into DNA during the S-phase of the cell cycle, can be detected immunochemically with monoclonal antibodies (mAbs) (10). This method has been applied to studies of cell cycle kinetics (51, frequency of sister chromatid exchanges (18,251, effects of drugs on DNA synthesis (71, and DNA replication and repair mechanisms (3,13,19).Denaturation of DNA is an essential step in current procedures for the exposure of DNA-incorporated BrdUrd to anti-BrdUrd mAbs, which recognize that base in denatured DNA, but not in native, duplex DNA. This denaturation step usually requires strong base or acid (e.g., NaOH or HC1) and often results in destruction of cell morphology and cellular antigens as well as cell loss and clumping. The development of a procedure by which both BrdUrd incorporation and antigen expression can be simultaneously detected enables the application of the BrdUrd assay to cytokinetic studies of cells which are concurrently phenotypically defined (1,14,23,24). We therefore sought to establish experimental conditions of DNA denaturation at which BrdUrd is detectable and cellular antigens are preserved. Several anti-BrdUrd mAb have been reported (6,8,10,14,20,23,28-30). Among these, the BU-1 mAb has a high binding affinity for BrdUrd and detects BrdUrd in DNA after deoxyribonuclease (DNase) treatment without harsh denaturation (8,9). DNase treatment was not successful with B44, another mAb (9).

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Section: Dnase Treatment Of Cellsmentioning

confidence: 99%

Detection of 5‐bromo‐2‐deoxyuridine (BrdUrd) incorporation with monoclonal anti‐brdurd antibody after deoxyribonuclease treatment

et al. 1993

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Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion

et al. 1992

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A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.

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A method for the prolonged culture of colonic epithelial cells

Whitehead

Eeden

1991

Journal of Tissue Culture Methods

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Rapid fluorometric detection of drug resistant tumour cells

Cited by 11 publications

References 18 publications

Detection of 5‐bromo‐2‐deoxyuridine (BrdUrd) incorporation with monoclonal anti‐brdurd antibody after deoxyribonuclease treatment

Detection of 5‐bromo‐2‐deoxyuridine (BrdUrd) incorporation with monoclonal anti‐brdurd antibody after deoxyribonuclease treatment

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion

A method for the prolonged culture of colonic epithelial cells

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