Quantifying apoptosis in banked human brains using flow cytometry

Juan Olano, Dwayne Wolf, Michael Keherly, Benjamin Gelman

Research output: Contribution to journalArticle

10 Citations (Scopus)

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
StatePublished - Nov 1996

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Flow Cytometry
Apoptosis
Brain
DNA Nucleotidylexotransferase
DNA Breaks
Streptavidin
Deoxyribonucleases
Fluorescein-5-isothiocyanate
In Situ Nick-End Labeling
Human Development
DNA Fragmentation
Encephalitis
Centrifugation
Brain Neoplasms
Neuroglia
Neurodegenerative Diseases
Paraffin
Sucrose
Cell Death
HIV

Keywords

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

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Neuroscience(all)

Cite this

Quantifying apoptosis in banked human brains using flow cytometry. / Olano, Juan; Wolf, Dwayne; Keherly, Michael; Gelman, Benjamin.

In: Journal of Neuropathology and Experimental Neurology, Vol. 55, No. 11, 11.1996, p. 1164-1172.

Research output: Contribution to journalArticle

Olano, Juan ; Wolf, Dwayne ; Keherly, Michael ; Gelman, Benjamin. / Quantifying apoptosis in banked human brains using flow cytometry. In: Journal of Neuropathology and Experimental Neurology. 1996 ; Vol. 55, No. 11. pp. 1164-1172.
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