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. New Method 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. Results 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. Comparison with Existing Method(s) 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.

Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes

“…The procedure was carried out essentially as described previously (Kaja et al, 2011; Kaja et al, 2012; Kaja et al, 2015a; Kaja et al, 2015b). Briefly, ONHAs were seeded onto 12 mm round poly-L-lysine coated glass coverslips (BD Biosciences, San Jose, CA) in a 24-well plate at a density of 7,500 cells.…”

“…We used chemically-induced, exogenously-applied oxidative stress as an in vitro insult to test the efficacy of the prototypic antioxidant, Trolox. We have previously validated the use of a standard oxidant, t BHP (Sigma Aldrich Corp.), for plate reader-based glioprotection studies using C6 astroglioma cells (Kaja et al, 2015b). …”

“…The MTT assay was performed essentially as described by us previously for C6 astroglia cells, HT-22 cells, and primary cortical neuron culture (Burroughs et al, 2012; Kaja et al, 2011; Kaja et al, 2015b). Briefly, the medium was aspirated from the cells and replaced with 100 μl of 1.2 mM MTT in HBSS with calcium and magnesium (Lonza, Walkersville, MD) supplemented with 10 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) at pH 7.3.…”

“…We optimized a custom LDH release assay for ONHAs from the previously developed and validated protocol for glioprotection using C6 astroglioma cells (Kaja et al, 2015b). Following 5 hr exposure to t BHP, 50 μl of cell culture supernatant were transferred to a non-sterile, clear 96-well well plate (Nunc, Thermo Fisher Scientific, Waltham, MA).…”

“…We have previously validated our custom LDH assay in C6 astroglioma cells (Kaja et al, 2015b). Herein, we show that the assay can be adapted readily to other cell types, including primary cells.…”