Quantifying apoptosis in banked human brains using flow cytometry

Juan P. Olano, Dwayne Wolf, Michael Keherly, Benjamin B. Gelman

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Fragmentation of genomic DNA, a major biochemical feature of programmed cell death (apoptosis), is easily detected when apoptosis is prevalent. In brain tissue apoptotic cells are usually scarce and detection requires more sensitive techniques. We describe a highly sensitive method to quantify apoptosis in frozen human brain tissue using flow cytometry. Nuclei from homogenized brain specimens were isolated to purity using discontinuous isopyknic centrifugation through 2.2 M sucrose. DNA strand breaks in apoptotic nuclei were conjugated with biotinylated-dUTP using terminal deoxynucleotidyl transferase (TdT) and tagged with streptavidin-conjugated FITC (TUNEL). Negative controls excluding the TdT step, and positive controls using DNAase pretreatment to create 3'-OH strand breaks were run in parallel. The proportion of nuclei with TdT-dependent labeling in adult brain specimens was <0.01% in 6 out of 7 specimens. In 3 fetal brains it averaged 0.86 ± 0.11%. Apoptotic cells were readily detected in 2 malignant glial neoplasms and in a patient with HIV encephalitis. Comparable frequencies of stained nuclei were present in adjacent specimens embedded in paraffin and labeled in situ. By screening millions of nuclei cytometry detected very rare apoptotic events, producing quantitative results using banked frozen brains. The method has potential applications to studies of human brain development, neurodegenerative diseases, and brain tumors.

Original languageEnglish (US)
Pages (from-to)1164-1172
Number of pages9
JournalJournal of Neuropathology and Experimental Neurology
Volume55
Issue number11
DOIs
StatePublished - Nov 1996

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Keywords

  • AIDS
  • Brain banking
  • Cell death
  • Isopyknic centrifugation
  • Nuclear isolation
  • TUNEL

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Neurology
  • Clinical Neurology
  • Cellular and Molecular Neuroscience

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