Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step.
Immunochemical detection of cells that incorporate 5-bromodeoxyuridine (BrdUrd) requires prior denaturation of DNA in situ to make BrdUrd binding sites accessible to the antibodies. A technique is described in which the DNA denaturation step is facilitated by a) prior dissociation of histones from DNA and b) the use of low ionic strength buffer in which the cells are suspended during heating. Dissociation of histones is achieved by cell treatment with 0.08N HCl at 0 degree C, which a) increases accessibility of DNA to propidium iodide (and following the denaturation to the antibodies); b) lowers stability of DNA to thermal denaturation; c) decreases differences between various cell types due to variability in chromatin structure; and d) ensures more complete DNA denaturation. Cell heating (80-95 degrees C) at low ionic strength (1 mM Na+) eliminates the need for formamide and results in extensive and rapid DNA denaturation. The method was applied in Friend leukemia, L1210 and HL-60 cell lines, and to bone marrow, experimental animal tumor and primary human tumor cells.
D000818:Animals; D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007939:Leukemia L1210 / Q000378:metabolism; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism; D051379:Mice; D009691:Nucleic Acid Denaturation; D009994:Osmolar Concentration; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D006657:Histones; D004247:DNA; D001973:Bromodeoxyuridine