Detection of Necrosis by Release of Lactate Dehydrogenase Activity

  • Francis Ka-Ming ChanEmail author
  • Kenta Moriwaki
  • María José De Rosa
Part of the Methods in Molecular Biology book series (MIMB, volume 979)


Apoptosis and necrosis are two major forms of cell death observed in normal and disease pathologies. Although there are many assays for detection of apoptosis, relatively few assays are available for measuring necrosis. A key signature for necrotic cells is the permeabilization of the plasma membrane. This event can be quantified in tissue culture settings by measuring the release of the intracellular enzyme lactate dehydrogenase (LDH). When combined with other methods, measuring LDH release is a useful method for the detection of necrosis. In this chapter, we describe the step-by-step procedure for detection of LDH release from necrotic cells using a microtiter plate-based colorimetric absorbance assay.

Key words

Programmed necrosis Necroptosis Membrane leakage Lactate dehydrogenase 



This work was supported by NIH grants (AI083497 and AI088502) to F. K-M. C. K. M. is supported by a postdoctoral fellowship from the Uehara Memorial Foundation, Japan. MJDR is supported by a CONICET fellowship from Argentina.


  1. 1.
    Schweichel JU, Merker HJ (1973) The morphology of various types of cell death in prenatal tissues. Teratology 7:253–266CrossRefGoogle Scholar
  2. 2.
    Moquin D, Chan FK (2010) The molecular regulation of programmed necrotic cell injury. Trends Biochem Sci 35:434–441PubMedCrossRefGoogle Scholar
  3. 3.
    Challa S, Chan FK (2010) Going up in flames: necrotic cell injury and inflammatory diseases. Cell Mol Life Sci 67:3241–3253PubMedCrossRefGoogle Scholar
  4. 4.
    Kono H, Rock KL (2008) How dying cells alert the immune system to danger. Nat Rev Immunol 8:279–289PubMedCrossRefGoogle Scholar
  5. 5.
    Chipuk JE, Green DR (2005) Do inducers of apoptosis trigger caspase-independent cell death? Nat Rev Mol Cell Biol 6:268–275PubMedCrossRefGoogle Scholar
  6. 6.
    Chan FK, Shisler J, Bixby JG, Felices M, Zheng L, Appel M, Orenstein J, Moss B, Lenardo MJ (2003) A role for tumor necrosis factor receptor-2 and receptor-interacting protein in ­programmed necrosis and antiviral responses. J Biol Chem 278:51613–51621PubMedCrossRefGoogle Scholar
  7. 7.
    Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, Chan FK (2009) Phosphorylation-driven assembly of the RIP1-RIP3 complex ­regulates programmed necrosis and virus-induced inflammation. Cell 137:1112–1123PubMedCrossRefGoogle Scholar
  8. 8.
    Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1:489–495PubMedCrossRefGoogle Scholar
  9. 9.
    Burd JF, Usategui-Gomez M (1973) A colorimetric assay for serum lactate dehydrogenase. Clin Chim Acta 46:223–227PubMedCrossRefGoogle Scholar
  10. 10.
    Korzeniewski C, Callewaert DM (1983) An enzyme-release assay for natural cytotoxicity. J Immunol Methods 64:313–320PubMedCrossRefGoogle Scholar
  11. 11.
    Decker T, Lohmann-Matthes ML (1988) A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods 115:61–69PubMedCrossRefGoogle Scholar
  12. 12.
    Cory AH, Owen TC, Barltrop JA, Cory JG (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 3:207–212PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Francis Ka-Ming Chan
    • 1
    • 2
    Email author
  • Kenta Moriwaki
    • 1
    • 3
  • María José De Rosa
    • 1
    • 3
  1. 1.Immunology and Virology Program, Department of Pathology, Center for AIDS ResearchUniversity of Massachusetts Medical SchoolWorcesterUSA
  2. 2.Diabetes and Endocrinology Research CenterUniversity of Massachusetts Medical SchoolWorcesterUSA
  3. 3.The Diabetes and Endocrinology Research CenterUniversity of Massachusetts Medical SchoolWorcesterUSA

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