Cellular Senescence: Review and Reagent Selection Guide

Science Note

[Jan. 16, 2023]                                                                                                                                                                                                                       Previous Science Note
The Drivers of Cellular Senescence

Cellular senescence is a complex biological process that is influenced by several key factors: DNA damage, telomere shortening, oxidative stress, oncogene activation, and so on. These factors collectively contribute to the complex process of cellular senescence, which acts as a double-edged sword - protective in preventing cancer growth, but also contributing to aging and age-related diseases.

Apoptotic stress causes mtDNA release during senescence and drives the SASP
Click here for the original article: Stella Victorelli, et. al., Nature, 2023.

Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype
Click here for the original article: Mate Maus, et. al., Nature, 2023.
Genome-wide CRISPR activation screening in senescent cells reveals SOX5 as a driver and therapeutic target of rejuvenation
Click here for the original article: Yaobin Jing, et. al., Cell Stem Cell, 2023.

Point of Interest
- Some mitochondrial outer membrane permeabilization (MOMP) requires BAX and BAK macropores.
- These macropores allow the release of mitochondrial DNA (mtDNA) into the cytosol.
- Cytosolic mtDNA in turn activates the cGAS-STING pathway, a key regulator of the SASP.  
- Inhibition of MOMP in vivo reduces inflammatory markers and improves healthspan in aged mice.
 

Point of Interest
- Vascular and hemolytic injury trigger iron accumulation, which causes senescence and promotes fibrosis.
- Senescent cells persistently accumulate iron, even after the increase in extracellular iron has subsided.
- Cells exposed to various types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes.
- The high levels of labile iron fuel the generation of reactive oxygen species and the SASP.  
Point of Interest
- CRISPRa screening identifies a comprehensive set of rejuvenators against senescence.
- Activation of SOX5 initiates a rejuvenation program via epigenetic remodeling.
- SOX5 activation leads to the stimulation of the HMGB2 enhancer, resulting in subsequent geroprotective effects. 
- Gene therapy using only SOX5 has the potential to promote the regeneration of aged knee joints.
Related Techniques
           Cellular senescence detection SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples.
           Ferrous ion (Fe2+) detection FerroOrange and Mito-FerroGreen
           Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
           Lysosomal function Lysosomal Acidic pH Detection Kit-Green/Red and Green/Deep Red NEW
           Mitochondrial superoxide detection mtSOX Deep Red
           Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit / MT-1 MitoMP Detection Kit
           Oxygen consumption rate assay Extracellular OCR Plate Assay Kit
           Antibody/Protein labeling: quick and high recovery Fluorescein, Biotin, and Peroxidase Labeling Kit - NH2
The products listed above are currently on special promotional offer.
Related Applications

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
 NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay KitLactate Assay Kit-WST

 


 

Assessing Cellular Senescence

Open / Close the Article

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

Open / Close the Article

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
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 Reports2017, 18(9), 2148.
Inhibition of SETD8
(Methyltransferase)
Oxidative phosphorylation↑,  ribosome synthesis↑
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”Oncotarget2016, 7(11), 12286.
Atg7 knockout mouse
(Satellite cells)
Autophagy inhibition SA-ß-Gal, P15, p16, p21, γ-H2AX ROS↑, mitochondrial membrane potential
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.
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.
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.
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 Rep2015, 10(8), 1310.
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 Cells2016, 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 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)
Ex. 500 – 540 nm /
Em. 530 – 570 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
Substrate: SPiDER-ßGal
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 Live cells, 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# SG04 SG05 Deep Red: C548
Blue: C549
Green: N511
Red: N512

 

 

Lipid Accumulation (Lipotoxicity)

Open / Close the Article

 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

Open / Close the Article

 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

Open / Close the Article
 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

 

 

Product Classification

Product Classification