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Quantifying apoptosis in banked human brains using flow cytometry

J P Olano1, D Wolf, M Keherly

  • 1Department of Pathology, University of Texas Medical Branch, Galveston 77550, USA.

Journal of Neuropathology and Experimental Neurology
|November 1, 1996
PubMed
Summary
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Detecting apoptosis in human brain tissue is challenging due to scarce apoptotic cells. This study presents a highly sensitive flow cytometry method (TUNEL assay) for quantifying apoptosis in frozen brain samples.

Area of Science:

  • Neuroscience
  • Cell Biology
  • Biochemistry

Background:

  • Apoptosis, characterized by DNA fragmentation, is difficult to detect in scarce brain cells.
  • Sensitive methods are needed for quantifying apoptosis in human brain tissue.

Purpose of the Study:

  • To develop and validate a highly sensitive flow cytometry method for quantifying apoptosis in frozen human brain tissue.
  • To assess the prevalence of apoptosis in normal adult and fetal brain tissue, as well as in pathological conditions.

Main Methods:

  • Isolating nuclei from homogenized brain specimens using discontinuous isopyknic centrifugation.
  • Employing the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay to detect DNA strand breaks in apoptotic nuclei.
  • Utilizing flow cytometry to quantify TdT-dependent labeling, with negative and DNAase-treated positive controls.

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Main Results:

  • The method detected apoptosis in <0.01% of adult brain nuclei (6/7 specimens) but 0.86% in fetal brains.
  • Apoptotic cells were readily identified in malignant glial neoplasms and HIV encephalitis.
  • Quantitative results were obtained by screening millions of nuclei from banked frozen brains.

Conclusions:

  • Flow cytometry coupled with the TUNEL assay provides a highly sensitive method for quantifying apoptosis in frozen human brain tissue.
  • This technique can detect rare apoptotic events, offering valuable applications in studying brain development, neurodegenerative diseases, and tumors.