title: An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience.
abstract: BACKGROUND
  Quantification of lactate dehydrogenase (LDH) release is a widely accepted assay for the quantitative determination of cell viability and late-stage apoptosis. Major disadvantages of commercially available LDH assay kits include proprietary formulations, limited options for optimization and high cost, all resulting in limited reproducibility in research applications. Here, we describe a novel, custom LDH assay suitable in the context of plate reader-based screening of drug candidates for glioprotection, but with wide applicability to other cell types and experimental paradigms.
  
  
  METHODS
  We developed a novel and highly reproducible LDH release assay that is more cost-effective than commercially available assays with comparable performance. The assay was validated by assessing 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid antioxidant protection against tert-butylhydroperoxide-induced oxidative stress in C6 astroglioma cells. Assay performance was validated by direct comparison and compatible with other methods of measuring cellular viability, namely 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 6-carboxy-2', 7' dichlorodihydrofluorescein diacetate assays.
  
  
  RESULTS
  There was no statistically significant difference between results obtained with the novel custom assay and a commercially available assay CytoTox96® (Promega, Madison, WI).
  
  
  CONCLUSIONS
  The novel custom LDH release assay allows the reproducible quantification of cell viability and is highly cost-effective when compared to commercially available assays (approximately 25 times cheaper). In addition and in contrast to commercially available assays, the identification and detailed description of all assay components and procedures provide greater control over experimental conditions and design. We provide a detailed standard operating procedure permitting our novel assay to be readily adapted depending on experimental requirements.
authors: Vision Research Center, Department of Ophthalmology, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, USA. Electronic address: kajas@umkc.edu.
authors: Simon
authors: S
authors: Kaja
authors: Vision Research Center, Department of Ophthalmology, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, USA.
authors: Andrew J
authors: AJ
authors: Payne
authors: Vision Research Center, Department of Ophthalmology, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, USA.
authors: Tulsi
authors: T
authors: Singh
authors: Vision Research Center, Department of Ophthalmology, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, USA.
authors: Jasleen K
authors: JK
authors: Ghuman
authors: Vision Research Center, Department of Ophthalmology, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, USA.
authors: Erin G
authors: EG
authors: Sieck
authors: Vision Research Center, Department of Ophthalmology, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, USA.
authors: Peter
authors: P
authors: Koulen
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009488:Neurosciences; D017382:Reactive Oxygen Species / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide; D007770:L-Lactate Dehydrogenase
keywords: C6 glioma cell; Cell viability; Glia; Glioprotection; High-throughput screening; Lactate dehydrogenase; Plate reader