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

Summary

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.

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.

Introduction

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....

Protocol

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 embryo head above the eyes.
3. Remove the red tissue (heart and liver) and use them for genotyping if necessary.
4. Prepare 500 mL of a 1:1 mixture ....

Results

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 activation^23,25,38. 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 passages. W.......

Discussion

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 tissues^25,39,40. The fluorescence base.......

Materials

List of materials used in this article

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