Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes

Simon Kaja1, Andrew J. Payne1, Yuliya Naumchuk1, Peter Koulen2

1 Department of Ophthalmology and Vision Research Center, School of Medicine, University of Missouri–Kansas City, Kansas City, Missouri, 2 Department of Basic Medical Science, School of Medicine, University of Missouri–Kansas City, Kansas City, Missouri
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.26
DOI:  10.1002/cptx.21
Online Posting Date:  May, 2017
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Abstract

Drug discovery heavily relies on cell viability studies to assess the potential toxicity of drug candidates. L‐Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that catalyzes the concomitant interconversions of pyruvate to L‐lactate and NADH to NAD+ during glycolysis, and the reverse reactions during the Cori cycle. In response to cellular damage, induced by endogenous cellular mechanisms or as a result of exogenously applied insults, LDH is released from the cytoplasm into the extracellular environment. Its stability in cell culture medium makes it a well‐suited correlate for the presence of damage and toxicity in tissues and cells. We herein present protocols for a reproducible and validated LDH assay optimized for several cell types. In contrast to commercially available LDH assays, often associated with proprietary formulations and high cost, our protocols provide ample opportunities for experiment‐specific optimization with low variability and cost. © 2017 by John Wiley & Sons, Inc.

Keywords: cell viability; lactate dehydrogenase; plate reader‐based assays

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: LDH Release (LDHr) Assay
  • Alternate Protocol 1: Total LDH (LDHt) Assay
  • Support Protocol 1: Experimental Setup for Neuroprotection Studies Using Primary Cultured Rat Cortical Neurons
  • Support Protocol 2: Experimental Setup for Glioprotection Studies Using Primary Cultured Rat Optic Nerve Head Astrocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: LDH Release (LDHr) Assay

  Materials
  • Samples (typically undiluted supernatant from tissue culture experiments or cell/tissue lysate; see support protocols for examples on sample preparation) in wells of 96‐well microtiter plate (‘sample plate’)
  • Buffer A (see recipe)
  • Buffer B (see recipe)
  • MPMS supplement (see recipe)
  • Stop solution: 1 M acetic acid (see recipe)
  • 96‐well microtiter plates, clear
  • Orbital shaker
  • Plate reader (for measuring absorbance at 490 nm wavelength)

Alternate Protocol 1: Total LDH (LDHt) Assay

  Additional Materials (also see protocol 1Basic Protocol)
  • 10× lysis solution (see recipe)

Support Protocol 1: Experimental Setup for Neuroprotection Studies Using Primary Cultured Rat Cortical Neurons

  Materials
  • E18 rat cortical neurons (e.g., Burroughs et al., )
  • Poly‐amine‐ or poly‐D‐lysine/laminin‐coated 96‐well plates
  • Trolox (EMD Millipore, cat. no. 648471; CAS number 53188‐07‐1)
  • 70% tert‐butyl hydroperoxide (tBHP; Luperox; Sigma‐Aldrich, cat. no. 458139; CAS number 75‐91‐2)
  • Poly‐amine‐ (BD PureCoat; BD Biosciences, cat. no. 356717) or poly‐D‐lysine/laminin‐coated (BD BioCoat; BD Biosciences, cat. no. 356640) 96‐well plates

Support Protocol 2: Experimental Setup for Glioprotection Studies Using Primary Cultured Rat Optic Nerve Head Astrocytes

  Materials
  • Primary optic nerve head astrocytes prepared using standard protocols (Kaja, Payne, Naumchuk, et al., )
  • Trolox (EMD Millipore, cat. no. 648471; CAS number 53188‐07‐1)
  • 70% tert‐butyl hydroperoxide (tBHP; Luperox; Sigma‐Aldrich, cat. no. 458139; CAS number 75‐91‐2)
  • 96‐well flat‐bottom TPP plates (MidSci, cat. no. TP92096)
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Figures

Videos

Literature Cited

  Babson, A. L., & Babson, S. R. (1973). Kinetic colorimetric measurement of serum lactate dehydrogenase activity. Clinical Chemistry, 19, 766–769.
  Babson, A. L., & Phillips, G. E. (1965). A rapid colorimetric assay for serum lactic dehydrogenase. Clinica Chimica Acta, 12, 210–215. doi: 10.1016/0009‐8981(65)90032‐X.
  Burroughs, S. L., Duncan, R. S., Rayudu, P., Kandula, P., Payne, A. J., Clark, J. L., … Kaja, S. (2012). Plate reader‐based assays for measuring cell viability, neuroprotection and calcium in primary neuronal cultures. Journal of Neuroscience Methods, 203, 141–145. doi: 10.1016/j.jneumeth.2011.09.007.
  Capps, R. D., 2nd, Batsakis, J. G., Briere, R. O., & Calam, R. R. (1966). An automated colorimetric (tetrazolium salt) assay for serum lactate dehydrogenase. Clinical Chemistry, 12, 406–413.
  Decker, T., & Lohmann‐Matthes, M. L. (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. Journal of Immunological Methods, 115, 61–69. doi: 10.1016/0022‐1759(88)90310‐9.
  Kaja, S., Payne, A. J., Naumchuk, Y., Levy, D., Zaidi, D. H., Altman, A. M., … Koulen, P. (2015). Plate reader‐based cell viability assays for glioprotection using primary rat optic nerve head astrocytes. Experimental Eye Research, 138, 159–166. doi: 10.1016/j.exer.2015.05.023.
  Kaja, S., Payne, A. J., Patel, K. R., Naumchuk, Y., & Koulen, P. (2015). Differential subcellular Ca2+ signaling in a highly specialized subpopulation of astrocytes. Experimental Neurology, 265, 59–68. doi: 10.1016/j.expneurol.2014.12.014.
  Kaja, S., Payne, A. J., Singh, T., Ghuman, J. K., Sieck, E. G., & Koulen, P. (2015). An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience. Journal of Pharmacological and Toxicological Methods, 73, 1–6. doi: 10.1016/j.vascn.2015.02.001.
  Kendig, D. M., & Tarloff, J. B. (2007). Inactivation of lactate dehydrogenase by several chemicals: Implications for in vitro toxicology studies. Toxicology in Vitro, 21, 125–132. doi: 10.1016/j.tiv.2006.08.004.
  Nachlas, M. M., Margulies, S. I., Goldberg, J. D., & Seligman, A. M. (1960). The determination of lactic dehydrogenase with a tetrazolium salt. Analytical Biochemistry, 1, 317–326. doi: 10.1016/0003‐2697(60)90029‐4.
  Raabo, E. 1963. Determination of serum lactic dehydrogenase by the tetrazolium salt method. Scandinavian Journal of Clinical and Laboratory Investigation, 15, 233–238. doi: 10.3109/00365516309079738.
  Stoddart, M. J. (2011). Cell viability assays: Introduction. In M. J. Stoddart (Ed.), Mammalian cell viability: Methods and protocols (pp. i–xi). New York: Humana Press.
  Vega‐Avila, E., & Pugsley, M. K. (2011). An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. Proceedings of the Western Pharmacology Society, 54, 10–14.
Key References
  Burroughs et al. (2012). See above.
  Detailed description of primary neuronal culture for plate reader‐based assays.
  Kaja, Payne, Naumchuk, et al. (2015). See above.
  Detailed description of primary optic nerve head astrocyte culture for plate reader–based assays, including examples of kinetic analysis LDHr, etc.
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