ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 329 (2004) 35–42 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.02.007 Use of cellular glucose-6-phosphate dehydrogenase for cell quantitation: applications in cytotoxicity and apoptosis assays Robert H. Batchelor ¤ and Mingjie Zhou Molecular Probes, Inc., 29851 Willow Creek Road, Eugene, OR 97402, USA Received 30 September 2003 Available online 24 April 2004 Abstract A Xuorescence-based microplate assay was developed to quantify cell death based upon the measurement of glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD is a cytosolic enzyme and leaks from cells when plasma membrane integrity is compromised. In this assay, cell death is measured by correlating the activity of extracellular G6PD to the reduction of resazurin to the Xuorescent product, resoruWn, via a coupled-enzyme reaction. The coupled-enzyme reaction permits rapid signal ampliWcation from small amounts of G6PD, an advantage over assays based on resazurin alone. This assay is rapid, nontoxic, and amenable to high-through- put screening. The assay has a Z 0 factor of 0.78.  2004 Elsevier Inc. All rights reserved. Keywords: Fluorescence; Microplate; G6PD; Cell death; ResoruWn; Resazurin; High-throughput Cell death is an important phenomenon associated with many areas of biology, including cancer, develop- ment, and aging. Cell death can be measured by a variety of methods. Some methods rely on exclusion or uptake of small-molecule tracers, such as trypan blue [1], neutral red [2], propidium iodide [3], [ 3 H]thymidine, [ 125 I]iodode- oxyuridine, 5-bromodeoxyuridine, carboxyXuorescein diacetate [4], or [ 51 Cr] [5,6]. However, these assays either are not adaptable to high-throughput screening or require prelabeling of the cells, often with radioactive probes. To avoid these problems, a number of assays that measure the amount of cytoplasmic contents released from dying cells have been developed. During either necrosis or apoptosis, the integrity of the plasma mem- brane is compromised, permitting cytosolic elements to leak into the extracellular milieu. Techniques using acid [7] or alkaline [8] phosphatase and other enzymes as cellular markers have been described, but these assays often suVer from low and/or variable amounts of enzyme present in diVerent cell types. The most common release assay measures the reduction of tetrazolium salts such as 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H- tetrazolium-5-carboxanilide, or 2-[4-iodophenyl]-3-[4- nitrophenyl]-5-phenyltetrazolium chloride (INT) by lactate dehydrogenase (LDH) 1 [9,10]. LDH release assays have two main disadvantages: (1) the tetrazolium salts typically require organic solubilization before quantitation and (2) there is considerable background LDH activity in most serum-containing growth media, which increases the background and limits sensitivity of these assays. While background LDH activity can be avoided by minimizing the amount of serum in the medium, this may result in decreased cell viability at longer time points. Another common dye used to measure cellular reduc- tive power as an indicator of cell viability is Alamar blue [11], also known as resazurin [12]. The reduction of resa- zurin to resoruWn, predominantly by dehydrogenases ¤ Corresponding author. Fax: 1-541-984-5698. E-mail address: rob.batchelor@probes.com (R.H. Batchelor). 1 Abbreviations used: LDH, lactate dehydrogenase; G6PD, glucose- 6-phosphate dehydrogenase; G6P, glucose-6-phosphate; DMEM, Dulbecco’s modiWed Eagle’s medium; HBSS, Hank’s balanced salt solution; FBS, fetal bovine serum; INT, 2-[4-iodophenyl]-3-[4-nitro- phenyl]-5-phenyltetrazolium chloride.