Cellular Senescence: Review and Reagent Selection Guide

Science Note

[Sep. 10, 2024]                                                                                                                                                                                                                       Previous Science Note
Senescence and Lipid Droplet Accumulation are Involved in the Onset of Neurodegenerative Diseases.

Recent research on senescence is revealing that senescence is related to lipid droplet and lysosomal dysfunction, which impair cell function. Here are some of the papers showing that these processes leads to neurodegenerative diseases.

Cellular senescence, a state of irreversible growth arrest, is closely linked to neurodegeneration through the accumulation of damaged cells in the nervous system. Lipid droplets, which store excess lipids, can accumulate in aging or stressed cells, contributing to cellular dysfunction and exacerbating neurodegenerative processes. Lysosomal dysfunction plays a central role in both lipid accumulation and the removal of cellular waste, and its impairment leads to the accumulation of toxic substances that further drive neurodegeneration. Together, these mechanisms create a cycle of cellular damage that accelerates the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's.
Lipid accumulation drives cellular senescence in dopaminergic neurons
Click here for the original article: Taylor Russo, et. al., Aging, 2024.		 Senescent glia link mitochondrial dysfunction and lipid accumulation
Click here for the original article: China Byrns, et. al., Nature, 2024.
 

 

 BHLHE40/41 regulate microglia and peripheral macrophage responses associated with Alzheimer’s disease and other disorders of lipid-rich tissues
Click here for the original article: Anna Podleśny-Drabiniok, et. al., Nature Communications, 2024.

Point of Interest
- Parkinson's disease (PD) is associated with the loss of dopaminergic neurons, with genetic and environmental factors contributing to its progression.

- Mutations in the lysosomal enzyme β-glucocerebrosidase cause lipid accumulation that drives cellular senescence in dopaminergic neurons in PD.

- Lipid droplet aggregation and lysosomal dysfunction may trigger cellular senescence leading to neurodegeneration in PD.

Point of Interest
- Senescent glia in aging Drosophila brains originate from neuronal mitochondrial dysfunction and express AP1, a senescence-associated transcription factor.

- AP1+ senescent glia cause lipid accumulation in non-senescent glia and increase senescence markers.

- Targeting AP1 in senescent glia extends lifespan, but increases oxidative damage in the brain and neuronal mitochondrial function remains poor.

Point of Interest
- Alzheimer's disease risk genes influence macrophage and microglial responses in lipid-rich tissues such as the brain.

- DLAMs are macrophage subpopulations with similar transcriptional activation states found in aging brains and other diseased lipid-rich tissues.

- Targeting BHLHE40/41, transcriptional regulators of DALM, may improve cholesterol clearance and lysosomal function in Alzheimer's disease therapies.

 

 
Related Techniques
           Cellular senescence detection SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples
NEW SPiDER-βGal Blue for fixed cell and for multiple staining with immunostaining and other methods
           Lipid Droplet detection Lipid Droplet Assay Kit - Blue / Deep Red
           Lipid Droplet Staining Lipi-Blue/ Green/ Red/ Deep Red
           Lysosomal function Lysosomal Acidic pH Detection Kit-Green/Red and Green/Deep Red
           First-time autophagy research Autophagic Flux Assay Kit
           Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
           Mitochondrial superoxide detection MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
           Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit
           Glutathione Quantification GSSG/GSH Quantification Kit
Related Applications

Co-staining with Lipid droplet and SA-β-Gal in fixed cells


*Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)

1. A549 (2 x 104) cells were seeded onto µ-slide 8 well plates (ibidi) and cultured overnight in a 37°C CO2 incubator. 
2. The supernatant was removed, washed once with PBS, and fixed in 4% paraformaldehyde (PFA)/PBS solution for 30 minutes at room temperature. 
3. The supernatant was removed and the cells were washed once with PBS. 
4. 15 µM SPiDER Blue + 0.1 µM Lipi-Deep Red prepared in Assay buffer was added and incubated at 37°C for 30 min.
5. The supernatant was removed, washed once with PBS, and 200 µl of PBS was added and observed under a confocal laser microscope (60x magnification).

 

 

Imaging analysis of lipid droplet accumulation in senescent cells was performed using normal A549 cells (CTRL)  and cells induced senescence by Doxorubicin treatment (DOX). SA-β-Gal was detected as a senescence marker with Cellular Senescence Detection Kit - SPiDER Blue, and lipid droplets were detected with Lipi-Deep Red. As a result, the signal of Lipi-Deep Red was increased in SA-β-Gal-positive senescent cells.

 

[Detection conditions]

SPiDE Blue: 405 nm (Ex), 400–550 nm (Em), 1.0%, 600V
Lipi-Deep Red: 633 nm (Ex), 650–700 nm (Em), 1.0%, 650V

 

Multiple staining with oxidative stress-related markers using Doxorubicin-induced senescent cells（flow cytometry)

Using A549 cells induced to senescence by doxorubicin (DOX) and normal cells (CTRL), changes in oxidative stress-related markers in senescent cells were analyzed by flow cytometry with multiple staining. SA-βGal as a senescence marker was detected by Cellular Senescence Detection Kit - SPiDER Blue, total ROS as an oxidative stress marker was detected by ROS Assay Kit - Photo-oxidation Resistant DCFH-DA-, and γH2AX as a DNA damage marker was detected by DNA Damage Detection Kit - γH2AX-Red. As a result, total ROS and γH2AX were increased in SA-βGal-positive senescent cells, and the increase in oxidative stress-related markers associated with cellular senescence could be detected by multiple staining.


  Flow cytometry：SONY SA3800
　 SPiDER Blue: PacificBlue 　
    ROS Assay Kit: FITC
    γH2AX - Red: Cy3

＜Experimental Procedure>
 *Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)
 The detail procedure for this experiment, please refer to the product page: SPiDER Blue.

 

 

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
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)

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

Search word