Techniques to Induce and Quantify Cellular Senescence

Summary

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

Abstract

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated β-galactosidase activity (SA-β-gal); p16^INK4A, p53, and p21 levels; higher levels of DNA damage, including γ-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-β-gal, γ-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Introduction

Over half a century ago, Hayflick and colleagues described how untransformed cells proliferate in culture, but for only a finite period of time¹. Long-term culturing of human fibroblasts caused the cells to stop proliferating; however, they were metabolically active, and this was called cellular senescence. Senescence can be beneficial for inhibiting tumorigenesis, but it also can be detrimental, as it is thought to contribute to the loss of regenerative capacity that occurs with aging^2,3. Senescent cells have been shown to accumulate in tissues as humans age⁴ an....

Protocol

1. Inducing Replicative Senescence

1. Thaw low-passage human diploid fibroblasts (e.g., WI-38 and IMR-90) or other cell lines.
   NOTE: Here, human diploid fibroblasts were used, but these protocols can be used to assess senescence in other cell types, such as endothelial, epithelial, or mesenchymal stem cells. Culturing conditions may be optimized for the different cell types used due to different growth rates or growth conditions.
   NOTE: Cells should be proliferating and have a spindle morphology (see Figure 1 for a schematic and Figure 2 for examples). Ideally, the cells should have a Population Doubling....

Results

Figures 2 - 6 show representative results from SA-β-gal staining; staining for γ-H2AX and SAHF; assessment of protein levels of p16^INK4A, p21, and p53; and mRNA and protein levels of senescent-associated molecules. Increased SA-β-gal staining occurs with replicative and DNA damage-induced senescence. Also, observe the morphological changes that occur with senescence. Cells become enlarged and flat compared to the spindle appearance of proliferating fibroblasts........

Discussion

Here, we have described methods for replicative and DNA-damage induced senescence using human diploid fibroblasts. In addition, techniques for quantifying protein and mRNA levels of various senescence-associated proteins are included, as well as staining for SA-β-gal and for the DNA-damage marker γ-H2AX. These protocols can be widely used to assess senescent phenotypes both in vitro and in vivo, although many caveats exist for characterizing senescence in vivo²⁰

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Tris-glycine gels	Invitrogen	XP00160BOX
Acid-Phenol ChCl3	Ambion	AM9720
Alexa-Fluor 568 goat anti-mouse antibody	Invitrogen	A11031	1:300 dilution
Cell lifters	Corning Inc.	3008	Cell scraper
ECL anti-mouse HRP linked antibody	Amersham	NA931V
ECL Plus Western Blotting Substrate	Pierce	32132	ECL
DAPI	Molecular Probes	MP01306	stock 5 mg/mL in dH2O
GAPDH antibody	Santa Cruz	sc-32233	1:1,000 - 5,000 dilution
GlycoBlue	Ambion	AM9515
Histone H3 dimethyl K9 monoclonal antibody	Abcam	1220	1:500 dilution
Human IL-6 Quantikine ELISA assay	R&D systems	D6050
Human IL-8 Quantikine ELISA assay	R&D systems	D8000C
Human GROa Quantikine ELISA assay	R&D systems	DRG00
N-N-dimethylformamide	Sigma	D4551	DMF
p16 monoclonal antibody	BD Biosciences	51-1325gr	1:500 dilution
p21 monoclonal antibody	Millipore	05-345	1:750 dilution
p53 monoclonal antibody	Santa Cruz	sc-126	1:500 dilution clone DO-1
phospho-H2AX (Ser139) FITC conjugate antibody	Cell Signaling	9719	1:2,000 dilution
POWER SYBR-green PCR master mix	Applied Biosystems	4367659
Pre-stained molecular weight markers	Biorad	161-0374
ProLong Gold Antifade	Invitrogen	P36930
PVDF membrane	Thermo Scientific	88518
Senescence β-Galactosidase Staining Kit	Cell Signaling	9860
TRIzol	Ambion/Life Tech	10296028