SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

J Vis Exp. 2019 Jun 28:(148). doi: 10.3791/58133.

Abstract

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-β-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated β-galactosidase (SA-β-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-β-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-β-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-β-Gal, cell morphology and cell number.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Biological Assay*
  • Biomarkers / metabolism
  • Cellular Senescence*
  • Female
  • Humans
  • Lysosomes / metabolism
  • Mice
  • Pregnancy
  • beta-Galactosidase / metabolism*

Substances

  • Biomarkers
  • GLB1 protein, human
  • beta-Galactosidase