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.
Materials and Reagents
Equipment
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This work was supported by the German Research Foundation (SFB TRR43 and HA5741/1-2 to Christoph Harms), the Federal Ministry of Education and Research (01 EO 08 01) for funding of the Center for Stroke Research Berlin (project ‘SUMO and stroke’ to Christoph Harms), and Berlin Institute of Health, TRG7, TP1 to Christoph Harms.
References
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