Induction and Validation of Cellular Senescence in Primary Human Cells

Summary

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Abstract

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated β-galactosidase (SA-β-gal), the rate of DNA synthesis using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Introduction

In 1961, Hayflick and Moorhead reported that primary fibroblasts in culture lose their proliferative potential after successive passages¹. This process is caused by the sequential shortening of telomeres after each cell division. When telomeres reach a critically short length, they are recognized by the DNA-damage response (DDR) that activates an irreversible arrest of proliferation — also defined as replicative senescence. Replicative senescence is currently one of the many stimuli that are known to induce a state of permanent cell cycle arrest that renders cells insensitive both to mitogens and to apoptotic signals²

Protocol

1. General Preparation

1. Prepare D10 medium. Supplement DMEM medium-Glutamax with 10% FBS and 1% penicillin/streptomycin (Final concentration: 100 U/mL).
2. Prepare sterile PBS. Dissolve the tablets in water according to manufacturer’s instructions. Sterilize by autoclave.
3. Prepare 1x trypsin. Dilute 5 mL of Trypsin-Versene EDTA/10x 1:10 in 45 mL of sterile PBS.
   Note: Throughout the protocol, we use cell culture conditions that are closer to the physiological conditions for primary fibroblasts. This means that we incubate cells at 37 °C and 5% CO₂ as is normally done but using 5% O₂ instea....

Results

Enrichment of SA-β-gal staining in senescent fibroblasts

Β-galactosidase (β-gal) is a lysosomal enzyme that is expressed in all cells and that has an optimum pH of 4.0^25,26. However, during senescence, lysosomes increase in size and, consequently, senescent cells accumulate β-gal. The increased amounts of this enzyme make it possible to detect its activity even at a subopti.......

Discussion

The protocols explained here were optimized for human primary fibroblasts, particularly BJ and WI-38 cells. The protocols for replicative senescence, ionizing radiation and doxorubicin, have been successfully applied to other types of fibroblasts (HCA2 and IMR90) and in other cell types (namely neonatal melanocytes and keratinocytes or iPSC-derived cardiomyocytes) in our laboratory. However, adaptations for additional cell types can be optimized by adjusting some details such as the number of seeded cells, the methods an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM Media - GlutaMAX	Gibco	31966-047
Fetal Bovine Serum	Hyclone	SV30160.03
Penicillin-Streptomycin (P/S; 10,000 U/ml)	Lonza	DE17-602E
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	SC-202581
Nuclease-Free Water (not DEPC-Treated)	Ambion	AM9937
T75 flask	Sarstedt	833911002
Trypsin/EDTA Solution	Lonza	CC-5012
PBS tablets	Gibco	18912-014
1.5 ml microcentrifuge tubes	Sigma-Aldrich	T9661-1000EA
Corning 15 mL centrifuge tubes	Sigma-Aldrich	CLS430791
6-well plate	Sarstedt	83.3920
24-well plate	Sarstedt	83.3922
13mm round coverslips	Sarstedt	83.1840.002
Steriflip	Merck Chemicals	SCGP00525
Cesium137-source			IBL 637 Cesium-137γ-ray machine
UV radiation chamber			Opsytec, Dr. Göbel BS-02
Doxorubicin dihydrochloride	BioAustralis Fine Chemicals	BIA-D1202-1
Hydrogen peroxide solution	Sigma-Aldrich	7722-84-1
5-aza-2’-deoxycytidine	Sigma-Aldrich	A3656
SAHA	Sigma-Aldrich	SML0061
Sodium Butyrate	Sigma-Aldrich	B5887
X-gal (5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside)	Fisher Scientific	7240-90-6
Citric acid monohydrate	Sigma-Aldrich	5949-29-1
Sodium dibasic phosphate	Acros organics	7782-85-6
Potassium ferrocyanide	Fisher Scientific	14459-95-1
Potassium ferricyanide	Fisher Scientific	13746-66-2
Sodium Chloride	Merck Millipore	7647-14-5
Magnesium Chloride	Fisher Chemicals	7791-18-6
25% glutaraldehyde	Fisher Scientific	111-30-8,7732-18-5
16% formaldehyde (w/v)	Thermo-Fisher Scientific	28908
EdU (5-ethynyl-2’-deoxyuridine)	Lumiprobe	10540
Sulfo-Cyanine3 azide (Sulfo-Cy3-Azide)	Lumiprobe	D1330
Sodium ascorbate	Sigma-Aldrich	A4034
Copper(II) sulfate pentahydrate (Cu(II)SO4.5H2O)	Sigma-Aldrich	209198
Triton X-100	Acros organics	215682500
TRIS base	Roche	11814273001
LightCycler 480 Multiwell Plate 384, white	Roche	4729749001
Lightcycler 480 sealing foil	Roche	4729757001
Sensifast Probe Lo-ROX kit	Bioline	BIO-84020
UPL Probe Library	Sigma-Aldrich	Various
Human IL-6 DuoSet ELISA	R&D	D6050
Bio-Rad TC20	Bio-Rad
Counting slides	Bio-Rad	145-0017
Dry incubator	Thermo-Fisher Scientific	Heratherm
Dimethylformamide	Merck Millipore	1.10983
Parafilm 'M' laboratory film	Bemis	#PM992
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