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Mol Cells
Mol Cells
Molecules and Cells
1016-8478
0219-1032
Korean Society for Molecular and Cellular Biology

S1016-8478(24)00127-4
10.1016/j.mocell.2024.100102
100102
MiniResource
Brief guide to senescence assays using cultured mammalian cells
Kang Eunseok 1
Kang Chanhee 2
Lee Young-Sam 3
Lee Seung-Jae V. seungjaevlee@kaist.ac.kr
1⁎
1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, South Korea
2 School of Biological Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea
3 Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, South Korea
⁎ Corresponding author. seungjaevlee@kaist.ac.kr
23 7 2024
9 2024
23 7 2024
47 9 10010224 6 2024
6 7 2024
18 7 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Cellular senescence is a crucial biological process associated with organismal aging and many chronic diseases. Here, we present a brief guide to mammalian senescence assays, including the measurement of cell cycle arrest, change in cellular morphology, senescence-associated β-galactosidase (SA-β-gal) staining, and the expression of senescence-associated secretory phenotype (SASP). This work will be useful for biologists with minimum expertise in cellular senescence assays.

Keywords

Aging
Cell cycle arrest
Senescence-associated β-galactosidase
Senescence-associated secretory phenotype
Senescence
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pmcDEFINITION AND CLASSIFICATION OF CELLULAR SENESCENCE

Cellular senescence is defined as stable cell cycle arrest (Hayflick, 1961, Kumari and Jat, 2021), which is induced by damaging stimuli (Hernandez-Segura et al., 2018, Park et al., 2022). Senescent cells accumulate in various tissues during aging, contributing to organismal aging, and the onset of age-related diseases (Jo et al., 2023, Lee, 2022, López-Otín et al., 2023), although cells that exhibit senescence phenotypes also play a role in normal development and tissue remodeling (Muñoz-Espín et al., 2013, Yun et al., 2015). Studies have highlighted the significance of targeting senescent cells as a potential therapeutic strategy (Chaib et al., 2022, Kirkland and Tchkonia, 2020, Zhang et al., 2023) against age-related diseases and the vulnerability to death conferred by age-dependent functional decline (Kwon et al., 2023, Lee and Lee, 2022, López-Otín et al., 2023). Here, we describe a brief guide to senescence assays using cultured mammalian cells for biologists (Fig. 1).Fig. 1 General hallmarks of cellular senescence. Senescent cells display several key features, including increased mass of lysosomes and SA-β-gal staining, destabilization of the nuclear integrity, increased expression of CDK inhibitor genes, p16 and p21, and upregulation of the DNA damage response. In addition, senescent cells exhibit increased mitochondrial mass and ROS, and decreased mitochondrial membrane potential, enlarged and flattened morphology, and secretion of SASP. CDK, cyclin-dependent kinase; ROS, reactive oxygen species; SA-β-gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype.

Fig. 1

Several types of cellular senescence have been characterized. Replicative senescence refers to the gradual loss of proliferative capacity after a limited number of cell divisions. Consecutive cell divisions lead to telomere shortening, triggering DNA damage response, which induces cell cycle arrest by upregulating several cell cycle inhibitors, including p16INK4a and p21CIP1 (Harley et al., 1990, Muñoz-Espín and Serrano, 2014). Human dermal fibroblasts (Yang et al., 2022), human foreskin fibroblasts (Gire et al., 2004), and human lung fibroblasts such as IMR-90, MRC-5, and WI-38 (Ahmed et al., 2010; Gire et al., 2004; Marthandan et al., 2016) have been utilized as popular model cells for replicative senescence. Oncogene-induced senescence is another type of senescence triggered by the induction of various oncogenes, including oncogenic RAS, both in vitro and in vivo (Gorgoulis and Halazonetis, 2010; Serrano et al., 1997). Oncogene-induced senescence can be induced in human lung fibroblasts by infecting them with a lentivirus carrying H-RAS (Quijano et al., 2012). DNA-damaging agents, such as bleomycin, doxorubicin (Piegari et al., 2013; Robles and Adami, 1998; Ryu et al., 2023), and ionizing radiation (Hong et al., 2010), result in DNA damage-induced senescence (Chen et al., 2007a). Other stimuli including oxidative stress (Chen et al., 2007a, Chen et al., 2007b; Song et al., 2023) and mitochondrial dysfunction (Wiley et al., 2016) cause oxidative stress-induced senescence and mitochondrial dysfunction-associated senescence, respectively. Researchers need to properly choose the types of senescence and cells depending on the purpose of their experiments.

MEASUREMENT OF CELL CYCLE ARREST

The most well-known hallmark of senescence is cell cycle arrest. Cell cycle arrest is characterized by the upregulation of cyclin-dependent kinase (CDK) inhibitors p16INK4a and p21CIP1. Activation of two major pathways, p16INK4a/RB and p53/p21CIP1 axes, blocks the proliferation of cells, contributing to senescence. p16INK4a is encoded by the CDKN2A gene and p21CIP1 is encoded by CDKN1A gene. During senescence, the level of p16INK4a increases by various cellular stresses, and p21CIP1 is upregulated by p53 activation via phosphorylation, following DNA-damaging stimuli (González-Gualda et al., 2021, Rayess et al., 2012). The levels of these proteins are quantified by employing various techniques, including immunofluorescence, immunohistochemistry, and western blotting. mRNA levels of these senescence marker genes are measured using quantitative real-time PCR (qRT-PCR). Several studies provide information on antibodies and primer sequences commonly used to detect these senescence markers (González-Gualda et al., 2021, Victorelli et al., 2023). Other methods that measure growth arrest in senescent cells include cell cycle phase assay and 5-bromo-2′-deoxyuridine (BrdU) or 5-ethynyl-2′-deoxyuridine (EdU) incorporation (Kumari and Jat, 2021, Mao et al., 2012). The cell cycle phase assay measures the G1 and G2 phase arrest by analyzing DNA content. DNA-binding dyes, such as propidium iodide, 4,6-diamidino-2-phenylindole (DAPI), and Hoechst 33342, are commonly used for quantifying DNA content (Ligasová et al., 2023). BrdU or EdU incorporation assays measure the DNA synthesis rate by using BrdU or EdU, a thymidine or uridine analog. Senescent cells exhibit reduced incorporation of these nucleotides, which is caused by the loss of replicative ability (González-Gualda et al., 2021).

MORPHOLOGY ALTERATION IN SENESCENT CELLS

Cells undergo several structural aberrations during senescence, including enlarged and flattened morphology, and increased mass of several organelles such as lysosomes, mitochondria, and nucleus (Hernandez-Segura et al., 2018). The size of the cells and the mass of membranous organelles progressively increase during cellular senescence (Kim et al., 2010, Mitsui and Schneider, 1976). Increased cell size, in turn, contributes to cell cycle arrest that is associated with senescence (Neurohr et al., 2019). The size and granularity of the cells are quantified using microscopy and flow cytometry (McKinnon, 2018; Neurohr et al., 2019). Senescent cells display destabilization of the nuclear integrity conferred by the downregulation of lamin B1 (Freund et al., 2012, Vergnes et al., 2004), which serves as a biomarker of cellular senescence (Heckenbach et al., 2022, Hernandez-Segura et al., 2017). The levels of lamin B1 are measured with western blot and qRT-PCR assays (Freund et al., 2012).

SENESCENCE-ASSOCIATED β-GALACTOSIDASE STAINING

Lysosomal β-D-galactosidase cleaves β-linked terminal galactosyl residues (Kurz et al., 2000), and it displays maximal activity between pH 4.0 and 4.5 (Lee et al., 2006). β-galactosidase is accumulated along with the increased lysosomal content in senescent cells (Hwang and Song, 2023, Kurz et al., 2000) and is detectable even at pH 6.0 (Dimri et al., 1995). Cytochemical or histochemical senescence-associated β-galactosidase (SA-β-gal) activity is measured by using chromogenic β-gal substrate (5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) in buffer at pH 6.0 after cell fixation. The SA-β-gal activity is also quantified by detecting fluorescence after treating with fluorescein di-β-D-galactopyranoside (FDG) or 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG); these substrates for β-gal emit fluorescence (excitation wavelength: 488 nm, emission wavelength: 530 nm) upon cleavage by the enzyme (Debacq-Chainiaux et al., 2009, Plovins et al., 1994, Yang and Hu, 2004).

SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE ASSAYS

Senescent cells secrete interleukins, cytokines, and growth factors, collectively termed senescence-associated secretory phenotype (SASP). These SASP factors influence surrounding cells by regulating various cell-surface receptors and signal transduction pathways (Coppé et al., 2010, Junaid et al., 2022, Oh et al., 2022). Additionally, SASP plays an essential role in the pathophysiological activity of senescent cells (Hernandez-Segura et al., 2018). qRT-PCR and antibody arrays are commonly used for detecting SASP expression. Studies provide qRT-PCR primer sequences commonly used for measuring the mRNA levels of SASP genes in mouse and human cells (González-Gualda et al., 2021, Victorelli et al., 2023). Antibody arrays are used to quantitatively assess secreted SASP protein levels (Coppé et al., 2008).

ASSESSMENT OF ALTERED CELLULAR FEATURES FOLLOWING SENESCENCE

Accumulating DNA damage is a typical feature of cellular senescence. DNA damage response increases the signals for phosphorylation of the histone variant H2AX (γH2AX) (Mah et al., 2010). The formation of γH2AX foci and its colocalization with 53BP1 are commonly measured to detect DNA damage in senescent cells (Chen et al., 2007a; Mah et al., 2010; Oda et al., 2023). The comet assay, a single-cell gel electrophoresis, is also widely utilized for estimating DNA damages in senescence research (Gyori et al., 2014).

Another feature of cellular senescence is abnormal mitochondrial function. Senescent cells exhibit mitochondrial dysfunction characterized by increased mitochondrial mass, reactive oxygen species (ROS) production, and decreased mitochondrial membrane potential (Miwa et al., 2022, Passos et al., 2007). Fluorescent dyes, Mitotracker and MitoSOX, are used for measuring mitochondrial mass and mitochondrial ROS, respectively (Miwa et al., 2022, Mukhopadhyay et al., 2007). For measuring mitochondrial membrane potential, fluorescent lipophilic cationic dyes, including tetramethylrhodamine methyl ester (TMRM), tetramethylrhodamine ethyl ester (TMRE), rhodamine 123, JC-1, and DiOC6(3), are commonly used (Nicholls, 2018, Perry et al., 2011). Senescent cells display increased numbers and sizes of lysosomes, along with pH neutralization (Robbins et al., 1970, Tan and Finkel, 2023). The increased mass of lysosomes is detected using Lysotracker, a fluorescent dye commonly used for labeling acidic organelles (Ivanov et al., 2013).

CONCLUDING REMARKS

Here we briefly described the types of cellular senescence and popular methods for assaying senescence. While detailed protocols need to be modified depending on specific senescence and cell types, our manuscript will be useful for biologists with minimum expertise in senescence assays. For more comprehensive research, users need to refer to additional publications, in particular for in vivo senescence assays that are not described in this manuscript.

Funding and support

This work was supported by KAIST Stem Cell Center (A0801080001 to S-J.V.L.).

Author contributions

Seung-Jae V. Lee: Writing – review & editing, Writing – original draft. Young-Sam Lee: Writing – review & editing. Chanhee Kang: Writing – review & editing. Eunseok Kang: Writing – review & editing, Writing – original draft.

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank all Lee laboratory members for their helpful discussion and comments.

ORCID

Eunseok Kang: https://orcid.org/0009-0009-6617-0831

Chanhee Kang: https://orcid.org/0000-0003-4350-5706

Young-Sam Lee: https://orcid.org/0000-0002-4702-0127

Seung-Jae V. Lee: https://orcid.org/0000-0002-6103-156X
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