A short history of the initial application of anti–5‐BrdU to the detection and measurement of S phase

Abstract

Because this article is a bit of history rather than a scientific paper, we will depart from the formality of a journal article. This article has two purposes. The first is to give credit to two deceased individuals, Albert Castro, M.D., and Howard Gratzner, Ph.D. (1). Castro was the key person who was able to produce a polyclonal antibody that was specific for bromodeoxyuridine (BrdU). For 10 years, Gratzner took the lead in converting an idea into a functional assay that affected DNA analysis and led to the development of many other assays with similar methodologies. The second is to inform young scientists that (a) inventions or creations have utility in fields other than the purpose for which they were created; (b) it is possible to do useful work in an institution that certainly would not have been classified as one of the best; and (c) the presence of talented people who are willing to collaborate is invaluable.Scientifically, Gratzner is best known for the development of the first monoclonal antibody (mAb) to BrdU and iododeoxyuridine. This work was the culmination of previous efforts with Robert Leif to develop polyclonal antibodies, which detected BrdU with variable specificity. Collaborations with scientists at the Lawrence Livermore Laboratory encouraged the development of a widely used flow cytometric test for the simultaneous measurements of DNA and BrdU. The use of mAbs to halogenated pyrimidines was a significant scientific advance and currently is an integral tool for the analysis by flow and digital microscopy of the cell cycle.On a personal level, Gratzner's enthusiasm for science and his intelligence, curiosity, loyal interactions with fellow collaborators, and friendship are sorely missed by all who knew him. He was genuinely a warm and nice person who was liked by almost everyone with whom he came in contact (1).Castro was a jovial man who interacted well with his collaborators. He was generous and very knowledgeable. He ran an immunodiagnostics laboratory at the universities of Oregon and Miami and was an expert in the production of antibodies in animals. His warm smile, generous giving of his knowledge, common sense, enthusiasm for scientific research, and ability to get the job done were essential to this project.Because this is a story of events that happened approximately 30 years ago and the existing records are limited, the accuracy of this article is limited by our memory and those of our colleagues who have kindly helped with this project. Of course, this is our version of the events.Cell division and DNA replication are fundamental to biology and specifically to the biology of cancer. This article describes how a simple procedure for the detection and quantitation of DNA synthesis was developed. The precise determination of when S phase occurs in the cell cycle is of use to maximize the selective killing of tumor cells with cycle-specific chemotherapeutic drugs. Of greater significance, studies of the control of the cell cycle, including its detour into apoptosis, can provide extre...

Assessment of Cell Cycle Inhibitors by Flow Cytometry

“…For this purpose, a second dimension is needed by using BrdU incorporation as readout. (Leif et al, 2004). This step is the most critical and is achieved by different methods: 1.…”

“…2. Acid or alkali treatments, followed by a neutralization step (Leif et al, 2004). The denaturation of DNA can be varied with time, only partially allowing the use of DNA probes staining such as propidium iodide (PI) , 7-aminoactynomycin D, TOPRO-3, which interacts DNA requiring double helix conformation.…”

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

“…Then the cells that are actively replicating their DNA can be detected using anti-BrdU antibodies. One disadvantage of this method is that it requires denaturation of DNA to provide access for the BrdU antibody by harsh methods like acid or heat treatment, which may result in inconsistency among results 13,14 . Alternatively, we utilized a similar approach to monitor the actively dividing cells with a different thymidine analog, EdU.…”

Cyclin D1 Induction Preceding Neuronal Death via the Excitotoxic NMDA Pathway Involves Selective Stimulation of Extrasynaptic NMDA Receptors and JNK Pathway

“…Following the block for nonspecific binding (PBS containing 0.1% Triton X-100, 5% bovine serum albumin, 5% serum), cells were incubated with various primary antibodies. The monoclonal antibodies Ki-67 (BD Biosciences) or BrdU (Immunologicals Direct; 1: 1,000) were used as a marker expressed specifically during the cell cycle [36] or the S-phase only [37] , respectively. Other primary antibodies (1: 500) included: rabbit monoclonal anti-cyclin D1 (NeoMarker), mouse anti-NeuN, anti-microtubule-associated protein 2 (anti-MAP2), and anti-␤ -III-tubulin (all from Chemicon).…”