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An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience.

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.

journal_name

J Pharmacol Toxicol Methods

journal_title

Journal of pharmacological and toxicological methods

authors

Kaja S,Payne AJ,Singh T,Ghuman JK,Sieck EG,Koulen P

doi

10.1016/j.vascn.2015.02.001

subject

Has Abstract

pub_date

2015-05-01 00:00:00

pages

1-6

eissn

1056-8719

issn

1873-488X

pii

S1056-8719(15)00018-0

journal_volume

73

pub_type

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