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Biology

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

Published: June 28th, 2019

DOI:

10.3791/58133

1Department of Molecular Medicine and the Center on Aging, The Scripps Research Institute, 2Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 3Institute on the Biology of Aging and Metabolism, University of Minnesota
* These authors contributed equally

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells.

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.

Cellular senescence was described for the first time by Leonard Hayflick and Paul Moorhead, who showed that normal cells had a limited ability to proliferate in culture1. Senescent cells fail to proliferate despite the presence of nutrients, growth factors and lack of contact inhibition, but remain metabolically active2. This phenomenon is known as replicative senescence and was mainly attributed to the telomere shortening, at least in human cells3. Further studies have shown that cells can also be induced to undergo senescence in response to other stimuli, such as oncogenic stress (oncogene induc....

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Animal use was approved by the Scripps Florida Institutional Animal Care and Use Committee.

1. Generation of senescent murine embryonic fibroblast (MEF) – 12-15 days

  1. Isolate wild type and Ercc1-/- MEFs from pregnant female mice at embryonic day 13 (E13) as described previously33.
    NOTE: All following steps are carried out in a tissue culture hood under aseptic conditions and using sterile instruments.
  2. Resect the embr.......

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SA-β-Gal activity can be detected in cells that are induced to senesce by various ways from replicative exhaustion, genotoxic and oxidative stress, to oncogene activation23,25,38. In the current model using Ercc1-deficient mouse embryonic fibroblast cells, normoxic growth conditions (20% O2) were sufficient to induce cell senescence after cultivating them for a few passag.......

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SA-β-Gal is a well-defined biomarker for cellular senescence originally discovered by Dimri et al. (1995) showing that senescent human fibroblasts have increased activity of SA-β-Gal when assayed at pH 623 compared to proliferating cells. Meanwhile, in vitro and in vivo assay for SA-β-Gal have been established for different cell types and tissues25,39,40. The fluorescence base.......

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This work was supported by NIH Grants AG043376 (Project 2 and Core A, PDR; Project 1 and Core B, LJN) and AG056278 (Project 3 and Core A, PDR; and Project 2, LJN) and a grant from the Glenn Foundation (LJN).

....

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Name Company Catalog Number Comments
DMEM  Corning 10-013-CV medium
Ham's F10 Gibco 12390-035 medium
fetal bovine serum Tissue Culture Biologics 101 serum
1x non-essential amino acids Corning 25-025-Cl amino-acids
bafilomycin A1  Sigma B1793 lysosomal inhibitor
C12FDG Setareh Biotech 7188 b-Gal substrate
Hoechst 33342  Life Technologies H1399 DNA intercalation agent
17DMAG Selleck Chemical LLC 50843 HSP90 inhibitor
InCell6000 Cell Imaging System GE Healthcare High Content Imaging System

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