Simon Kaja1, Andrew J Payne2, Tulsi Singh2, Jasleen K Ghuman2, Erin G Sieck2, Peter Koulen2. 1. 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. 2. Vision Research Center, Department of Ophthalmology, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, USA.
Abstract
INTRODUCTION: 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). DISCUSSION: 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.
INTRODUCTION: 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). DISCUSSION: 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: Stephanie L Burroughs; R Scott Duncan; Parvathi Rayudu; Prasanthi Kandula; Andrew J Payne; Julie L Clark; Peter Koulen; Simon Kaja Journal: J Neurosci Methods Date: 2011-09-24 Impact factor: 2.390
Authors: S Kaja; R S Duncan; S Longoria; J D Hilgenberg; A J Payne; N M Desai; R A Parikh; S L Burroughs; E V Gregg; D L Goad; P Koulen Journal: Neuroscience Date: 2010-11-11 Impact factor: 3.590
Authors: Simon Kaja; Andrew J Payne; Yuliya Naumchuk; Deborah Levy; Danish H Zaidi; Alexa M Altman; Saba Nawazish; Jasleen K Ghuman; Bryan C Gerdes; Mark A Moore; Peter Koulen Journal: Exp Eye Res Date: 2015-06-03 Impact factor: 3.467
Authors: Maria Madalena Costa Sobral; Tiago Gonçalves; Zita E Martins; Christine Bäuerl; Erika Cortés-Macías; Maria Carmen Collado; Isabel M P L V O Ferreira Journal: Toxins (Basel) Date: 2022-01-01 Impact factor: 4.546