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Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures   

Edited by
Soyun Kim
Reviewed by
Anonymous reviewer
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In this protocol

Original research article

A brief version of this protocol appeared in:
The Journal of Neuroscience
Aug 2016

Abstract

The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in supernatants of cell cultures serve as a practicable tool for quantification of cellular injury (Koh and Choi, 1987; Bruer et al., 1997). Lactate dehydrogenase (LDH) is such a ubiquitously expressed cytosolic enzyme, which is very stable due to a very long protein half-life (Hsieh and Blumenthal, 1956; Koh and Cotman, 1992; Koh et al., 1995).

Keywords: LDH assay, Primary neurons, Cell damage, LDH release, Cell culture, Cell death, Survival, Oxygen-glucose deprivation, Glutamate toxicity

Background

LDH catalyzes the formation of lactate and Nicotinamidadenindinucleotid (NAD+) from pyruvate and reduced Nicotinamidadenindinucleotid (NADH) in a reversible biochemical reaction. NADH has an absorption on the wavelength of 340 nm. The basis of this kinetic LDH activity assay is the decrease of optical density at the specific wave length caused by a decrease of NADH. The amount of LDH in the supernatant is calculated using a standard enzyme solution with known LDH activity. Different cell densities or metabolic activation rates might be confounders; therefore normalization of LDH activity is recommended. This is achieved by assessment of LDH activity after outer cell membrane lysis that does not block LDH activity itself (‘full kill’ with 0.5% Triton-X). Finally, percentage of absolute LDH activity from LDH activity by full kill indicates the rate of damaged or dead cells in the cell culture well.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Freyer, D. and Harms, C. (2017). Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures. Bio-protocol 7(11): e2308. DOI: 10.21769/BioProtoc.2308.
  2. Donath, S., An, J., Lee, S. L., Gertz, K., Datwyler, A. L., Harms, U., Muller, S., Farr, T. D., Fuchtemeier, M., Lattig-Tunnemann, G., Lips, J., Foddis, M., Mosch, L., Bernard, R., Grittner, U., Balkaya, M., Kronenberg, G., Dirnagl, U., Endres, M. and Harms, C. (2016). Interaction of ARC and Daxx: A novel endogenous target to preserve motor function and cell loss after focal brain ischemia in mice. J Neurosci 36(31): 8132-8148.
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