Cellular Senescence: Review and Reagent Selection Guide DOJINDO LABORATORIES

Science Note

Uncovering Hidden Mechanisms of Cellular Senescence [Sep. 2, 2025]

New Insights Reveal How Senescence Spreads and Enlarged Cells Are Sustained
Aging is increasingly seen as a key factor behind many diseases, driving the search for ways to slow or prevent it. Recent work has shown that the protein HMGB1 can transmit senescence signals between cells and even to distant tissues, and that blocking this process supports tissue repair. Another study identified AP2A1 as a factor that keeps senescent cells enlarged, with its inhibition reversing aging traits. Together, these findings point to new possibilities for targeting the fundamental mechanisms of aging to improve health and longevity.

Reference

Related technique

Propagation of senescent phenotypes by extracellular HMGB1 is dependent on its redox state (Metabolism, 2025)
Summary: This study identifies redox-sensitive HMGB1 (ReHMGB1) as a novel driver of cellular senescence that spreads from one cell to its neighbors and through the bloodstream to distant organs. Blocking extracellular HMGB1 was shown to reduce systemic senescence and improve tissue regeneration, suggesting its potential as a therapeutic target for aging-related diseases.

Highlighted technique: The study evaluated cellular senescence using SA β gal staining to identify senescent cells and EdU incorporation to assess proliferative arrest. Expression levels of p16 and p21 were also measured as molecular markers of cell cycle inhibition.

AP2A1 modulates cell states between senescence and rejuvenation (Cellular Signalling, 2025)
Summary: Senescent cells are markedly larger than young cells, a feature that drives further aging and disease, yet the molecular basis of this enlarged architecture has remained unclear. This study reveals that AP2A1 reinforces integrin β1–mediated anchorage to maintain cell size, and its inhibition reverses senescence phenotypes, highlighting AP2A1 as a potential therapeutic target for aging-related conditions.

Highlighted technique: The study combined SA β gal staining with quantitative analysis of cell size to directly link senescence associated β galactosidase activity to the pronounced hypertrophy of senescent cells. This morphological and biochemical readout was then applied to assess the effects of AP2A1 knockdown on SA β gal positivity and cellular enlargement.

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
Lysosomal function	Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
First-time autophagy research	Autophagic Flux Assay Kit
Mitophagy detection	Mitophagy Detection Kit
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Endocytosis Detection	ECGreen-Endocytosis Detection
ATP Measurement	ATP Assay Kit-Luminescence
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)
> Senescent Cells Lose Mitochondrial Activity

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

Topics

What is Cellular Senescence?
Assessing Cellular Senescence
Reagents Selection Guide
Indicators Related to Cellular Senescence
Lipid Accumulation (Lipotoxicity)
Cell Cycle Arrest
Changes in Intracellular Metabolism

What is Senescence?

Lysosome Senescence in cell biology refers to a state of permanent cell cycle arrest in response to stresses such as DNA damage or oncogene activation. Senescent cells can be identified by several molecular markers. Representative examples include p53, a transcription factor involved in the DNA damage response; p16, a cyclin-dependent kinase inhibitor that enforces cell cycle arrest; and senescence-associated β-galactosidase (SA-βgal). These cells resist apoptosis and ferroptosis and secrete inflammatory factors, collectively known as the senescence-associated secretory phenotype (SASP). Although senescence plays protective roles such as tumor suppression in early stages, the accumulation of these cells over time promotes chronic inflammation and contributes to several age-related diseases. As immune-mediated clearance of senescent cells declines with age, understanding their biology is crucial for the development of therapies targeting ageing and its associated diseases.

Assessing Cellular Senescence

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

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: SG03(SG04) SPiDER Blue: SG07	SG05	Deep Red: C548 Blue: C549	Green: N511 Red: N512

Indicators Related to Cellular Senescence

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.
①	IMR90 (Human pulmonary fibroblasts)	Inhibition of SETD8 (Methyltransferase)	SA-ß-Gal, p16, p21, Nucleosome hypertrophy	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.
②	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.

Accumulation of Lipid Peroxides and Their Connection to Cellular Senescence and Mitochondria

Lipotoxicity is caused by intracellular lipid accumulation and is indicative of mitochondrial disfunction. Lipotoxicity accelerates the degenerative process of cellular senescence, influencing cancer development.

① Lipid Droplet Detection

② Mitochondria Dynamics

③ Senescence Marker Detection

④ Monitoring Mitochondrial Membrane Potential

⑤ Mitophagy Detection/Lysosome Detection

⑥ Lipid Peroxide Detection

⑦ Glucose Uptake Detection

⑧ Total ROS Detection/Mitochondrial Superoxide Detection

⑨ Cell Cycle Analysis

References

1. Clara, C. al., “Mitochondria: Are they causal players in cellular senescence?”, Biochimica et Biophysica Acta – Bioenergetics, 2015, 1847(11), 1373-1379.

2. Huizhen, Z. et al., “Lipidomics reveals carnitine palmitoyltransferase 1C protects cancer cells from lipotoxicity and senescence”, Journal of Pharmaceutical Analysis, 2020.

3. Xiaojuan, H. et al., “Astrocyte Senescence and Alzheimer’s Disease: A Review”, Front. Aging Neurosci., 2020.

4. Borén, J. et al., “Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation”, Cell Death Differ, 2012, 19(9), 1561-1570.

5. Na, L. et al., “Aging and stress induced β cell senescence and its implication in diabetes development”, Aging (Albany NY), 2019, 11(21), 9947–9959.

Cell Cycle Arrest

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

In aged cells, due to mitochondrial dysfunction, ATP is primarily generated through the anaerobic glycolysis pathway, leading to an increase in lactate production²⁾. DNA damage is one of the causes of mitochondrial dysfunction in cellular aging. The accumulation of DNA damage activates DNA repair mechanisms and increases NAD+ consumption. The decrease in NAD+ levels reduces SIRT1 activity, an important factor in maintaining mitochondrial function, leading to impaired mitochondrial function (inhibition of electron transfer → ATP production / reduction of NAD+ levels)^1),3).

Reference:

1. J. Wu, Z. Jin, H. Zheng and L. Yan, “Sources and implications of NADH/NAD+redox imbalance in diabetes and its complications”, Diabetes Metab. Syndr. Obes., 2016, 9, 145

2. Z. Feng, R. W. Hanson, N. A. Berger and A. Trubitsyn, “Reprogramming of energy metabolism as a driver of aging”, Oncotarget., 2016, 7(13), 15410.

3. S. Imai and L. Guarente, “NAD+ and sirtuins in aging and disease”, Trends in Cell Biology, 2014, 24(8), 464.