A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis

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Abstract

Apoptosis is an important mode of cell death under botn physiological and pathophysiological conditions. Numerous techniques are available for the study and quantitation of apoptosis in cell culture, but only few are useful when applied to complex tissues. Among these, the terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay remains the most widely used technique. However, its specificity and sensitivity for the detection of apoptosis remain controversial. We developed a technique consisting of staining live cells and tissues with Hoechst 33342 and the vital dye propidium iodide (PI), followed by fixation and the TUNEL reaction. We demonstrate excellent retention of PI in necrotic cells after fixation. We also examined the distribution of TUNEL staining among necrotic and apoptotic cells in various models of cell injury in vitro and in vivo. We show that the sensitivity of the TUNEL varied between 61 and 90% in the models examined. The specificity exceeded 87% in all models but fell to 70% when a predominantly necrotic injury was induced. This novel and simple method will permit the determination of indices of sensitivity and specificity for the TUNEL assay in other tissues and experimental conditions.

Original languageEnglish
JournalAmerican Journal of Physiology - Cell Physiology
Volume284
Issue number5 53-5
StatePublished - May 1 2003

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Transferases
Labeling
Apoptosis
Sensitivity and Specificity
Propidium
Tissue
Assays
Staining and Labeling
Wounds and Injuries
Cell death
Cell culture
Cell Death
Coloring Agents
Cell Culture Techniques
Cells

Keywords

  • Ischemia
  • Necrosis
  • Propidium iodide
  • Two-photon microscopy

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

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title = "A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis",
abstract = "Apoptosis is an important mode of cell death under botn physiological and pathophysiological conditions. Numerous techniques are available for the study and quantitation of apoptosis in cell culture, but only few are useful when applied to complex tissues. Among these, the terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay remains the most widely used technique. However, its specificity and sensitivity for the detection of apoptosis remain controversial. We developed a technique consisting of staining live cells and tissues with Hoechst 33342 and the vital dye propidium iodide (PI), followed by fixation and the TUNEL reaction. We demonstrate excellent retention of PI in necrotic cells after fixation. We also examined the distribution of TUNEL staining among necrotic and apoptotic cells in various models of cell injury in vitro and in vivo. We show that the sensitivity of the TUNEL varied between 61 and 90{\%} in the models examined. The specificity exceeded 87{\%} in all models but fell to 70{\%} when a predominantly necrotic injury was induced. This novel and simple method will permit the determination of indices of sensitivity and specificity for the TUNEL assay in other tissues and experimental conditions.",
keywords = "Ischemia, Necrosis, Propidium iodide, Two-photon microscopy",
author = "Katherine Kelly and Sandoval, {Ruben M.} and Kenneth Dunn and Bruce Molitoris and Pierre Dagher",
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TY - JOUR

T1 - A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis

AU - Kelly, Katherine

AU - Sandoval, Ruben M.

AU - Dunn, Kenneth

AU - Molitoris, Bruce

AU - Dagher, Pierre

PY - 2003/5/1

Y1 - 2003/5/1

N2 - Apoptosis is an important mode of cell death under botn physiological and pathophysiological conditions. Numerous techniques are available for the study and quantitation of apoptosis in cell culture, but only few are useful when applied to complex tissues. Among these, the terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay remains the most widely used technique. However, its specificity and sensitivity for the detection of apoptosis remain controversial. We developed a technique consisting of staining live cells and tissues with Hoechst 33342 and the vital dye propidium iodide (PI), followed by fixation and the TUNEL reaction. We demonstrate excellent retention of PI in necrotic cells after fixation. We also examined the distribution of TUNEL staining among necrotic and apoptotic cells in various models of cell injury in vitro and in vivo. We show that the sensitivity of the TUNEL varied between 61 and 90% in the models examined. The specificity exceeded 87% in all models but fell to 70% when a predominantly necrotic injury was induced. This novel and simple method will permit the determination of indices of sensitivity and specificity for the TUNEL assay in other tissues and experimental conditions.

AB - Apoptosis is an important mode of cell death under botn physiological and pathophysiological conditions. Numerous techniques are available for the study and quantitation of apoptosis in cell culture, but only few are useful when applied to complex tissues. Among these, the terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay remains the most widely used technique. However, its specificity and sensitivity for the detection of apoptosis remain controversial. We developed a technique consisting of staining live cells and tissues with Hoechst 33342 and the vital dye propidium iodide (PI), followed by fixation and the TUNEL reaction. We demonstrate excellent retention of PI in necrotic cells after fixation. We also examined the distribution of TUNEL staining among necrotic and apoptotic cells in various models of cell injury in vitro and in vivo. We show that the sensitivity of the TUNEL varied between 61 and 90% in the models examined. The specificity exceeded 87% in all models but fell to 70% when a predominantly necrotic injury was induced. This novel and simple method will permit the determination of indices of sensitivity and specificity for the TUNEL assay in other tissues and experimental conditions.

KW - Ischemia

KW - Necrosis

KW - Propidium iodide

KW - Two-photon microscopy

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