Open AccessThermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): Critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram
Author(s) -
Hoy C. A.,
Seamer L. C.,
Schimke R. T.
Publication year - 1989
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990100608
Subject(s) - denaturation (fissile materials) , bromodeoxyuridine , dna , staining , flow cytometry , fluorescence , biology , biophysics , microbiology and biotechnology , chemistry , biochemistry , cell growth , genetics , quantum mechanics , nuclear chemistry , physics
Significant inter‐ and intraexperimental variations of the relative antibromodeoxyuridine fluorescence were found during measurement of DNA synthesis rates using flow cytometric analysis of 5‐bromodeoxyuridine (BrdUrd)‐labeled cells with an anti‐BrdUrd antibody. Fluctuations in other endpoints associated with levels of denaturation (integrity of DNA and cell size) were also observed to vary widely among samples that were otherwise thought to have been treated identically. Therefore, the denaturation step has been carefully re‐examined, and several critical factors were identified that influence the denaturation and subsequent binding of the anti‐BrdUrd to the labeled DNA. These factors include cell density, volume of water, and pH of the sample during heating. Appropriate adjustments are now included in the protocol, resulting in more consistent anti‐BrdUrd measurements in the face of routine (and sometimes necessary) experimental variations.