Advanced search
1 file | 5.16 MB

Methods for distinguishing apoptotic from necrotic cells and measuring their clearance

Dmitri Krysko (UGent) , Tom Vanden Berghe (UGent) , Eef Parthoens (UGent) , Katharina D'Herde (UGent) and Peter Vandenabeele (UGent)
(2008) METHODS IN ENZYMOLOGY. 442. p.307-341
Author
Organization
Abstract
Three major morphological types of cell death can be distinguished: type I (apoptotic cell death), type 11 (autophagic cell death), and type III (necrotic cell death). Details of the pathways of apoptotic and autophagic cell death have been described, and distinct biochemical markers have been identified. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic parameters (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). Moreover, a confounding factor is that apoptotic cells in the absence of phagocytosis proceed to secondary necrosis, which has many morphological features of primary necrotic cells. Secondary necrotic cells have already gone through an apoptotic stage, and so it is generally advisable in cell death research to perform time kinetics of cell death parameters. This chapter concentrates on methods that can distinguish apoptosis from necrosis on three different levels (morphological, biochemical, and analysis of cell-cell interactions) and emphasizes that only a combination of several techniques can correctly characterize cell death type. First, we describe analysis of apoptotic versus necrotic morphology by time-lapse microscopy, flow fluorocytometry, and transmission electron microscopy. We also discuss various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow fluorocytometry), cellular markers such as DNA fragmentation (flow fluorocytometry), caspase activation, Bid cleavage, and cytochrome c release (Western blotting). Next, we describe how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin 18. Finally, we discuss cell-cell interactions during cell death and describe a quantitative method for examining dead cell clearance by flow fluorocytometry. A selection of techniques that can be used to study internalization mechanisms used by phagocytes to engulf dying cells is also presented, such as scanning and transmission electron microscopy and fluorescence microscopy.

Downloads

  • (...).pdf
    • full text
    • |
    • UGent only
    • |
    • PDF
    • |
    • 5.16 MB

Citation

Please use this url to cite or link to this publication:

Chicago
Krysko, Dmitri, Tom Vanden Berghe, Eef Parthoens, Katharina D’Herde, and Peter Vandenabeele. 2008. “Methods for Distinguishing Apoptotic from Necrotic Cells and Measuring Their Clearance.” Ed. Roya Khosravi-Far, Zahra Zakeri, Richard A Lockshin, and Mauro Piacentini. Methods in Enzymology 442: 307–341.
APA
Krysko, Dmitri, Vanden Berghe, T., Parthoens, E., D’Herde, K., & Vandenabeele, P. (2008). Methods for distinguishing apoptotic from necrotic cells and measuring their clearance. (R. Khosravi-Far, Z. Zakeri, R. A. Lockshin, & M. Piacentini, Eds.)METHODS IN ENZYMOLOGY, 442, 307–341.
Vancouver
1.
Krysko D, Vanden Berghe T, Parthoens E, D’Herde K, Vandenabeele P. Methods for distinguishing apoptotic from necrotic cells and measuring their clearance. Khosravi-Far R, Zakeri Z, Lockshin RA, Piacentini M, editors. METHODS IN ENZYMOLOGY. 2008;442:307–41.
MLA
Krysko, Dmitri, Tom Vanden Berghe, Eef Parthoens, et al. “Methods for Distinguishing Apoptotic from Necrotic Cells and Measuring Their Clearance.” Ed. Roya Khosravi-Far et al. METHODS IN ENZYMOLOGY 442 (2008): 307–341. Print.
@article{431717,
  abstract     = {Three major morphological types of cell death can be distinguished: type I (apoptotic cell death), type 11 (autophagic cell death), and type III (necrotic cell death). Details of the pathways of apoptotic and autophagic cell death have been described, and distinct biochemical markers have been identified. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic parameters (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). Moreover, a confounding factor is that apoptotic cells in the absence of phagocytosis proceed to secondary necrosis, which has many morphological features of primary necrotic cells. Secondary necrotic cells have already gone through an apoptotic stage, and so it is generally advisable in cell death research to perform time kinetics of cell death parameters. This chapter concentrates on methods that can distinguish apoptosis from necrosis on three different levels (morphological, biochemical, and analysis of cell-cell interactions) and emphasizes that only a combination of several techniques can correctly characterize cell death type. First, we describe analysis of apoptotic versus necrotic morphology by time-lapse microscopy, flow fluorocytometry, and transmission electron microscopy. We also discuss various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow fluorocytometry), cellular markers such as DNA fragmentation (flow fluorocytometry), caspase activation, Bid cleavage, and cytochrome c release (Western blotting). Next, we describe how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin 18. Finally, we discuss cell-cell interactions during cell death and describe a quantitative method for examining dead cell clearance by flow fluorocytometry. A selection of techniques that can be used to study internalization mechanisms used by phagocytes to engulf dying cells is also presented, such as scanning and transmission electron microscopy and fluorescence microscopy.},
  author       = {Krysko, Dmitri and Vanden Berghe, Tom and Parthoens, Eef and D'Herde, Katharina and Vandenabeele, Peter},
  editor       = {Khosravi-Far, Roya and Zakeri, Zahra and Lockshin, Richard A and Piacentini, Mauro},
  isbn         = {9780123743121},
  issn         = {0076-6879},
  journal      = {METHODS IN ENZYMOLOGY},
  language     = {eng},
  pages        = {307--341},
  title        = {Methods for distinguishing apoptotic from necrotic cells and measuring their clearance},
  url          = {http://dx.doi.org/10.1016/S0076-6879(08)01416-X},
  volume       = {442},
  year         = {2008},
}

Altmetric
View in Altmetric
Web of Science
Times cited: