Chemiluminescence-based BrdU ELISA to measure DNA synthesis James R. Hawker Jr. * Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A&M University System Health Science Center, Medical Research Building, Rm 206A, 702 SW H. K. Dodgen Loop, Temple, TX 76504, USA Received 11 January 2002; received in revised form 4 September 2002; accepted 2 October 2002 Abstract We describe a simple, sensitive, nonradioactive, relatively rapid and relatively inexpensive protocol to measure DNA synthesis in cultured cells by a chemiluminescent bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA). We show that it exhibits similar sensitivity and activity as traditional 3 H-thymidine incorporation assays and a commercial chemiluminescent BrdU ELISA kit when tested in commonly used cell lines, such as NIH 3T3 cells, mink lung epithelial cells (Mv1LU), and baby hamster kidney (BHK-21) fibroblasts. This assay also exhibits a wider dynamic range than colorimetric BrdU ELISA methods. Besides being a viable, nonradioactive alternative to 3 H-thymidine incorporation assays, our BrdU ELISA is less expensive than a commercial chemiluminescent BrdU ELISA kit. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Bromodeoxyuridine; Thymidine; DNA synthesis; ELISA; Cell culture 1. Introduction Incorporation of radioactive thymidine is a widely used method to quantify cell proliferation and/or initiation of DNA synthesis by exogenous growth factors, hormones, drugs, or other agents in cultured cells (Corps and Brown, 1993). However, issues associated with the costs and safety of the use and disposal of radioactive materials has led to the search for viable nonradioactive alternatives to measure cell proliferation or DNA synthesis in cultured cells. Several alternative nonradioactive methods have been devised to measure cell proliferation, such as cell- mediated reduction of tetrazolium dyes (Mosmann, 1983), assay of cellular acid phosphatase activity (Connolly et al., 1986; Yang et al., 1996), staining with crystal violet (Gillies et al., 1986), or staining of cell-associated protein (Tuszynski and Murphy, 1990). Similarly, the development of monoclonal antibodies directed against bromodeoxyuridine (BrdU), a chemical analog of thymidine, has led to the design of methods to measure DNA synthesis both in vivo (Raza et al., 1985) and in cultured cells by both immunocytochemistry (counting labeled nuclei) or by a BrdU ELISA (Magaud et al., 1988; Muir et al., 1990; Portsmann et al., 1985) in which the amount of BrdU incorporated into cultured cells in 0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0022-1759(02)00437-4 * H. L. Moffitt Cancer Center, MRC-DRDIS, 12902 Magnolia Dr, Tampa, FL 33612. Tel.: +1-813-979-6755; fax: +1-813-979- 6748. E-mail address: hawkerjr@moffitt.usf.edu (J.R. Hawker). www.elsevier.com/locate/jim Journal of Immunological Methods 274 (2003) 77 – 82