Cellular Senescence: Review and Reagent Selection Guide

Why is senescent cell detection important?

Cellular senescence leads to irreversible cell cycle arrest, metabolic dysfunction, and increased secretion of inflammatory factors, contributing to tissue deterioration and its strong association with cancer and neurodegenerative diseases. This page introduces the latest findings on how senescence contributes to disease progression and the underlying mechanisms that drive the formation of senescent cells.

Science Note

Senescence Driven by Mitochondrial Alterations [Mar. 5, 2025] 

Cellular senescence is closely linked to mitochondrial dysfunction, which drives aging and disease through metabolic shifts, oxidative stress, and pro-inflammatory signaling. This Science Note shows the latest findings on mitochondrial mechanisms that promote cellular senescence.

Apoptotic stress causes mtDNA release during senescence and drives the SASP
Stella Victorelli, et. al., Nature, 2023.
Apoptosis is triggered by widespread mitochondrial outer membrane permeabilization (MOMP), whereas in cellular senescence, sublethal minority MOMP occurs in a subset of mitochondria, releasing mtDNA into the cytosol and inducing SASP-driven inflammation.

Highlighted technique: When apoptotic stress increases the permeability of the mitochondrial outer membrane, cytochrome c is released into the cytoplasm, making it a key indicator of MOMP. In this study, MOMP is assessed by co-staining the mitochondrial outer membrane protein TOM20 and cytochrome c, followed by high-resolution analysis.

Mitochondrial fatty acid oxidation drives senescence
Shota Yamauchi, et. al., Science Advances, 2024.
DNA damage triggers the phosphorylation of BNIP3, a mitochondrial membrane protein, leading to an increase in cristae and enhanced FAO. This, in turn, promotes histone acetylation and induces the expression of the senescence marker p16.

Highlighted technique: This study assessed senescence using multiple markers, including p16 expression and SA-βgal activity. While other markers changed, γH2AX did not, reinforcing the widely accepted notion that no single marker defines senescence. Thus, multiple indicators are needed to assess senescence.

Senescent glia link mitochondrial dysfunction and lipid accumulation
China Byrnsn, et. al., Nature, 2024.
In the Drosophila brain, AP1+ senescent glia arise from neuronal mitochondrial dysfunction, promote lipid accumulation in non-senescent glia, and increase senescence markers. While suppressing AP1+ activity extends lifespan, it also exacerbates oxidative damage in the brain, leaving neuronal mitochondrial dysfunction unresolved, possibly due to reduced lipid accumulation.

Highlighted technique: In recent years, many studies have reported a link between senescence and Lipid droplets (LD). LDs are detected with BODIPY 493/503 as used in this paper. We also offer Lipi-Dye, a dye specifically designed for LD detection with high intracellular retention. With its range of color variations, it can be a valuable tool for researchers studying LD.

Previous Science Note
Related Techniques (click to open/close)
Target Kit & Probes
Cellular senescence detection SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples.
Blue cellular senescence detection dye for fixed cells,  SPiDER Blue
Mitophagy Detection Mitophagy Detection Kit
Oxygen Consumption Rate(OCR) Detection Extracellular OCR Plate Assay Kit
Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Lipid Droplet Staining Lipi-Blue/ Green/ Red/ Deep Red
Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Mitochondrial superoxide detection MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit
Apoptosis detection in multiple samples Annexin V Apoptosis Plate Assay Kit
Cell proliferation/ cytotoxicity assay Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST
 Application Note  (click to open/close)
  > Metabolic shift to glycolysis in senescenct cells


 

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471


Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

 

  
     

 
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Assessing Cellular Senescence

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What is Cellular Senescence?

 Cellular senescence was reported by Hayflick in 1981. It was discovered when pulmonary fibroblasts slowed down their proliferation and eventually ended in cell death after cell passaging had been performed for more than 8 months. Subsequent studies have revealed that cellular senescence is caused not only by telomere length reduction, but also by external factors such as oncogene activation, oxidative stress, and DNA damage.
The induction and control mechanisms of cellular senescence – in which genetic and external factors are intricately involved – have yet to be fully elucidated. However, it has been suggested that the process is closely related to cancer and various age-related diseases, inspiring large amounts of active research into the topic. The development of drugs that eliminate senescent cells in the body (senolytic drugs) is also attracting the attention of researchers as a possible strategy to extend healthy life expectancy.





 
 
 
 
 
 
 
 Cellular senescence is controlled by various factors such as cell type and physiological conditions, such as oxidative stress. None of the individual biomarkers that have been identified so far have been deemed to be specific to senescent cells. Therefore, it is desirable to determine and confirm cellular senescence using multiple indicators.
 Common detection indicators for assessing cellular senescence include features related to cell cycle progression (DNA synthesis, p16/p21 expression, etc.), features related to morphology (of the cell, nucleus, nucleolus, etc.), SA-ß-Gal activity, DNA damage, oxidative stress (ROS), telomere length, inflammatory cytokines (senescence-associated secretory phenotype (SASP)), and more.

 

< Video Seminar >
“Recent Findings of Cellular Senescence Studies and Analysis Method”

Chapters:
0:00 What is Cellular Senescence ?
5:00 Senescence Studies and Drug discovery
12:30 Methods of Senescence Detection and Analysis

 

 

 

Indicators Related to Cellular Senescence

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Correlation map of related indicators

 Research into apoptosis, necrosis, autophagy, and cellular senescence is very important for understanding the intracellular functions that control cell survival and death.
Recently, various fields have given particular attention to cellular senescence due to the recent discoveries of SASP (a known cancer-causing factor) and senescence-related phenomena in stem cell research.

 This correlation map shows the relationship between various intracellular indicators, resulting from cellular senescence. This information is based on currently available information. Please refer to the table with cited references below as reference for your experiments. The table lists the cell type, the method of senescence induction used, the senescence markers measured, and the variables affected by senescence in each reference for the map.

  Cell Senescence induction Senescence marker (s) Responding variable (s) Reference
① IMR90
(Human pulmonary fibroblasts)		 Several passages in culture SA-ß-Gal, p16, p21, Nucleosome hypertrophy Expression of SETD8↓,  H4K20me1↓, oxidative phosphorylation↑, ribosome synthesis↑ H. Tanaka, S. Takebayashi, A. Sakamoto, N. Saitoh, S. Hino and M. Nakao, “The SETD8/PR-Set7 Methyltransferase Functions as a Barrier to Prevent Senescence-Associated Metabolic Remodeling.”, Cell Reports, 2017, 18(9), 2148.
Inhibition of SETD8
(Methyltransferase)		 Oxidative phosphorylation↑,  ribosome synthesis↑
② Senescent mouse satellite cell
eletal muscle progenitor cells)		 – SA-ß-Gal, p16 Autophagy activity↓, ROS↑, mitochondrial membrane potential↓ L. Garcia-Prat, M. Martinez-Vicente and P. Munoz-Canoves, “Autophagy: a decisive process for stemness”, Oncotarget, 2016, 7(11), 12286.
Atg7 knockout mouse
(Satellite cells)		 Autophagy inhibition SA-ß-Gal, P15, p16, p21, γ-H2AX ROS↑, mitochondrial membrane potential↓
③ Rat fibroblast model of type 2 diabetes – SA-ß-Gal, p21, p53, γ-H2AX NADP+/ NADPH↓(resistance to oxidative stress↓), NADPH oxidase↑(ROS↑) M. Bitar, S. Abdel-Halim and F. Al-Mulla, “Caveolin-1/PTRF upregulation constitutes a mechanism for mediating p53-induced cellular senescence: implications for evidence-based therapy of delayed wound healing in diabetes”, Am J Physiol Endocrinol Metab., 2013, 305(8), E951.
④ IMR90
(Human pulmonary fibroblasts)		 Ethidium bromide (inhibition of mtDNA) + pyruvate deficiency SA-ß-Gal NAD+/NADH C. Wiley, M. Velarde, P. Lecot, A. Gerencser, E. Verdin, J. Campisi, et. al., “Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype”, Cell Metab., 2016, 23(2), 303.
⑤ MDA-MB-231
(Human breast cancer cells)		 X-ray irradiation + inhibition of cell cycle-related factor (securin) expression SA-ß-Gal Lactate↑, LDH activity↑, (glycolysis↑) E. Liao, Y. Hsu, Q. Chuah, Y. Lee, J. Hu, T. Huang, P-M Yang & S-J Chiu, “Radiation induces senescence and a bystander effect through metabolic alterations.”, Cell Death Dis., 2014, 5, e1255.
⑥ MEF
(Mouse Embryonic Fibroblast)		 Overexpression of oncogenes,several passages in culture, transcription factor overexpression(E2F1) SA-ß-Gal, p16, p21, Nucleosome hypertrophy Ribosome RNA↑, p53↑ K. Nishimura, T. Kumazawa, T. Kuroda, A. Murayama, J. Yanagisawa and K. Kimura, “Perturbation of Ribosome Biogenesis Drives Cells into Senescence through 5S RNP-Mediated p53 Activation”, Cell Rep. 2015, 10(8), 1310.
⑦ Mouse tail fibroblast 2 months old, 22 months old, p16 knockout (22 months old) SA-ß-Gal, p14, p16 NAD+↓, SIRT3↓ M. J. Son, Y. Kwon, T. Son and Y. S. Cho, “Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells”, Stem Cells. 2016, 34(12), 2840.

 

 

 

Reagent Selection Guide

Dojindo offers four types of kits and reagents that can be selected according to the evaluation method and purpose of cell senescence.

Product Cellular Senescence Detection Kit – SPiDER-ßGal, SPiDER Blue Cellular Senescence Plate Assay Kit – SPiDER-ßGal Cell Cycle Assay Solution Deep Red / Blue Nucleolus Bright Green / Red
Detection Fluorescence Fluorescence Fluorescence Fluorescence
Wavelength
(Ex/Em)		 [SPiDER-ßGal]
Ex. 500–540 nm/Em. 530–570 nm
[SPiDER Blue]
Ex. 350-450 nm/Em. 400-500 nm		 Ex. 535 nm / Em. 580 nm Deep Red: Ex. 633-647 nm /
Em. 780/60 nm
Blue: Ex. 405-407 nm /
Em. 450/50 nm		 Green: Ex. 513 nm /
Em. 538 nm
Red: Ex. 537 nm /
Em. 605 nm
Target SA-ß-gal activity SA-ß-gal activity Nucleus Changes in the nucleolus
Detection
Method		 Imaging, Flow cytometry
Substrate: SPiDER-ßGal, SPiDER Blue		 Plate assay
Substrate: SPiDER-ßGal		 Flow cytometry Imaging Detection of the nucleolus by RNA-staining reagent
Instrument Fluorescence microscope, FCM Fluorescence microplate reader FCM Fluorescence microscope
Sample SPiDER-ßGal: Live or fixed cells
SPiDER Blue: Fixed cells
(Tissue: some examples from published articles using SG02)		 Live cells
(lysis of live cells)		 Live cells, fixed cells Fixed cells
Best for Those who have difficulty quantifying data or performing multiple staining with X-gal Those who process multiple samples
Those who are evaluating senescent cells for the first time Small size package (20 tests) is available		 Those who wish to evaluate using indicators other than SA-ß-Gal Those who wish to evaluate using indicators other than SA-ß-Gal
Examples of reports using nucleolus as an indicator are available on the product page
data
Item# SPiDER-ßGal: SG04
SPiDER Blue: SG07		 SG05 Deep Red: C548
Blue: C549		 Green: N511
Red: N512

 

Lipid Accumulation (Lipotoxicity)

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 Lipotoxicity, which is caused by excessive lipid accumulation in non-adipose tissue cells, is thought to be involved in cancer, diabetes, heart failure, and obesity. It has been shown that lipid accumulation in cells causes cellular senescence and mitochondrial dysfunction. The figure below shows the changes in various indices caused by excessive lipid accumulation.

For more information click here or image below

 

 

 

Cell Cycle Arrest

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 Irreversible cell cycle arrest is one of the phenomena that characterize cellular senescence. p16, p21, p53, and pRB (phosphorylated retinoblastoma protein) are known as representative protein markers. The activation/upregulation of these proteins are used as indicators of cellular senescence. These marker proteins are known to be tumor suppressors and regulate the cell cycle mainly through two pathways (p16Ink4a-RB and p53-p21CIP1).

Doxorubicin (DOX) is known as an anticancer drug that acts in the G2/M phase of the cell cycle to arrest cell proliferation and induce cellular senescence (see the figure below in center). Below are the results of an experiment in which DOX was added to A549 cells. As a result, changes in SA-ß-Gal expression, cell cycle progression, and mitochondrial membrane potential were observed.

Changes in Intracellular Metabolism

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 It is well known that senescent cells maintain high metabolic activity despite their reduced proliferative capacity. In general, senescent cells show a decrease in NAD+, an increase in lactate efflux, and a decrease in AMP/ATP ratio. This is due to conversion from oxidative phosphorylation to aerobic glycolysis and mitochondrial dysfunction, in addition to activation of the glycolytic system.
Changes in intracellular metabolism are thus closely related to cellular senescence. Therefore, these changes in intracellular metabolism are very important – not only as indicators of cellular senescence, but also in clinical and basic research targeting cellular senescence.
Our webpage on intracellular metabolism provides maps focusing on senescence-associated changes in intracellular metabolism, such as SIRT1-related changes in NAD+ levels, and cells that have become senescent due to DNA damage.
(Please click on the "Senescence" tab in the link)
 
 
 

 

 

 

Related Scientific Information

Autophagy

 

 

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