Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal anti-BrdUrd antibody after deoxyribonuclease treatment.
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 anti-BrdUrd 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 III 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 III offers a simple approach to quantitation of apoptotic cells, in which an endogenous endonuclease is activated.
Takagi
S
S
Department of Pharmacology, University of Massachusetts Medical School, Worcester 01655.
McFadden
M L
ML
Humphreys
R E
RE
Woda
B A
BA
Sairenji
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D017209:Apoptosis; D001854:Bone Marrow Cells; D001973:Bromodeoxyuridine / Q000276:immunology / Q000378:metabolism*; D002051:Burkitt Lymphoma / Q000473:pathology; D002460:Cell Line; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology*; D004720:Endonucleases / Q000494:pharmacology; D005092:Exonucleases / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D005559:Formamides / Q000494:pharmacology; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000473:pathology; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D005559:Formamides; C031066:formamide; D004247:DNA; D004720:Endonucleases; D005092:Exonucleases; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases; D001973:Bromodeoxyuridine