Cellular Senescence: Review and Reagent Selection Guide

Science Note

Systemic Signals in Aging and Rejuvenation [May 14, 2025]

Cellular senescence spreads through tissues via secreted factors, contributing to tissue dysfunction, chronic inflammation, and impaired regeneration. This Science Note introduces recent insights into the in vivo spread of senescence and highlights emerging evidence that rejuvenating factors can also spread, suggesting new avenues for modulating aging and restoring tissue function.

Mitochondrial DNA released by senescent tumor cells enhances PMN-MDSC-driven immunosuppression through the cGAS-STING pathway (Immunity, 2025)
Summary: Senescent cells, including those undergoing therapy-induced senescence, actively shed mitochondrial DNA (mtDNA) into the tumour microenvironment via extracellular vesicles. This mtDNA is taken up by polymorphonuclear myeloid-derived suppressor cells, enhancing their immunosuppressive activity through cGAS-STING-NF-κB signalling and promoting tumour progression.

Highlighted technique: To assess mtDNA transfer from tumour senescent cells, extracellular vesicles (EVs) were isolated and confirmed to be mtDNA enriched. Their uptake by BM-MDSCs led to intracellular mtDNA accumulation, whereas EV-suppressed senescent cells failed to induce mtDNA accumulation during co-culture with BM-MDSCs.

Related technique Cellular Senescence Detection, Exosome Labeling Dye

Hepatocellular senescence induces multi-organ senescence and dysfunction via TGFβ (Nature Cell Biology, 2024)
Summary: Hepatocyte senescence in acute severe liver disease induces senescence in extrahepatic organs, contributing to multi-organ failure. This systemic spread is mediated by the TGFβ pathway and its inhibition can prevent secondary organ dysfunction.

Highlighted technique: his study used a mouse model enabling liver-specific and time-controlled deletion of MDM2, a suppressor of p53. MDM2 removal led to p53 accumulation and p21 upregulation in liver cells, allowing investigation of how liver senescence affects other organs.

Related technique Cellular Senescence Detection

Small extracellular vesicles from young plasma reverse age-related functional declines by improving mitochondrial energy metabolism (Nature Aging, 2024)
Summary: Small extracellular vesicles (sEVs) from young mice reverse age-related changes in aged mice by improving molecular, cellular and physiological functions, including lifespan extension and reduced senescence. These effects are mediated by miRNA, which improves mitochondrial function and metabolic processes in aged tissues.

Highlighted technique: In this study, researchers collected sEVs from the blood plasma of young mice and injected them into older mice to observe the effects. They examined lifespan, cognitive function and mitochondrial activity, which is often impaired in ageing, to show the reversal of signs of ageing in the mice.

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
Exosome Monitoring	ExoSparkler Exosome Membrane Labeling Kit-Green / Red / Deep Red
Exosome Isolation	ExoIsolator Exosome Isolation Kit and Isolation Filter
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial Staining	MitoBright LT Green / Red / Deep Red
Oxygen Consumption Rate(OCR) Detection	Extracellular OCR Plate 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 I (click to open/close)
> Senescent Cells Lose Mitochondrial Activity

The senescent cell detection dye SPiDER Blue (SG07) and the mitochondrial membrane potential (MMP) dye MT-1 (MT13) were used to stain human microglial cells. Microscopy revealed that, compared to control cells, senescence-induced cells showed reduced MMP and increased SPiDER Blue fluorescence, reflecting elevated SA-β-Gal activity.

*This data was kindly provided by Dr. Supriya D. Mahajan, Department of Medicine, Jacobs School of Medicine & Biomedical Sciences.

1.　Seed human microglia cells into a dish and incubate in an incubator set at 37 ℃ and equilibrated with 95% air and 5% CO₂.
2.　Dilute the MT-1 Dye (1:1000) in the cell culture medium.
3.　Add MT-1 working solution to cells.
4.　Incubate the cells for 30 minutes in an incubator set at 37 ℃ and equilibrated with 95% air and 5% CO₂.
5.　Discard the supernatant and wash the cells with HBSS twice.
6.　Add 4% PFA/ PBS solution to the cells and incubate at room temperature for 30 minutes.
7.　Discard the 4% PFA / PBS solution and wash the cells with PBS.
8.　Add 15 µmol/l Spider Blue working solution and incubate at 37°C for 30 minutes.
9.　Remove the working solution, and wash the cells with PBS.
10. Add Imaging Buffer solution and observe the cells under a fluorescence microscope.

Application Note II (click to open/close)
> Increased Senescence in Aged Adipose Tissue

Frozen liver adipose tissue sections from 8-week-old and 35-week-old mice were stained with senescence detection probes SPiDER Blue (SG07) and SPiDER-βGal (SG02). Confocal microscopy revealed a marked increase in fluorescence intensity only in the 35-week-old samples, indicating an age-associated accumulation of senescent cells in older tissue.

1. 8-week-old and 35-week-old mouse liver adipose tissue (frozen sections) samples were prepared on glass slides.
2. After washing once with PBS, 200 µl of 4% paraformaldehyde (PFA)/PBS solution was added and fixed at room temperature for 30 minutes.
3. The supernatant was removed and washed once with PBS.
4. Add 200 µl of 15 µM SPiDER Blue and 15 µM SPiDER-βGal prepared in Assay buffer and incubate at 37°C for 2 hours.
5. The supernatant was removed and washed once with PBS.
6. Add 1 drop of encapsulant (ProLong Glass Antifade Mountant, Thermo) and encapsulate with cover glass.
7. Observed under a confocal laser microscope (40x magnification).

[Detection conditions]
SPiDER Blue: 405 nm (Ex), 400–500 nm (Em), 2.0%, 700V
SPiDER-βGal: 488 nm (Ex), 500–600 nm (Em), 1.0%, 600V

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.