Cellular Senescence - Amerigo Scientific

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.

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-β-Gal activity, DNA damage, oxidative stress (ROS), telomere length, inflammatory cytokines (senescence-associated secretory phenotype (SASP)), and more.

Indicators Related to Cellular Senescence

Research into apoptosis, necrosis, autophagy, and cellular senescence is very important for understanding the intracellular functions that control cell survival and death.

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.

Indicators Related to Cellular Senescence

	Cell	Senescence induction	Senescence marker (s)	Responding variable (s)	Reference
①	IMR90 (Human pulmonary fibroblasts)	Several passages in culture	SA-β-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-β-Gal, p16, p21, Nucleosome hypertrophy	Oxidative phosphorylation↑, ribosome synthesis↑
②	Senescent mouse satellite cell (eletal muscle progenitor cells)	-	SA-β-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-β-Gal, P15, p16, p21, γ-H2AX	ROS↑, mitochondrial membrane potential↓
③	Rat fibroblast model of type 2 diabetes	-	SA-β-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-β-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-β-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-β-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-β-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.

Products

CAT. No.	Product Name	Size
SG03-10	Cellular Senescence Detection Kit - SPiDER-βGal	10 assays
R252-10	ROS Assay Kit -Highly Sensitive DCFH-DA-	100 tests
L256-10	Lactate Assay Kit-WST	50 tests
N509-10	NAD/NADH Assay Kit-WST	100 tests
N510-10	NADP/NADPH Assay Kit-WST	100 tests
N511-10	Nucleolus Bright Green	60 nmol
MT13-10	MT-1 MitoMP Detection Kit	1 set
D676-10	DAPGreen - Autophagy Detection	5 nmol

Cellular Senescence Reagent Selection

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

Product Name	Cellular Senescence Detection Kit - SPiDER-βGal	Cellular Senescence Plate Assay Kit - SPiDER-βGal	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-β-gal activity	SA-β-gal activity	Nucleus	Changes in the nucleolus
Detection Method	Imaging Substrate: SPiDER-βGal	Plate assay Substrate: SPiDER-βGal	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-β-Gal	Those who wish to evaluate using indicators other than SA-β-Gal; Examples of reports using nucleolus as an indicator are available on the product page
Data
CAT. No.	SG03	SG05	Deep Red: C548 Blue: C549	Green: N511 Red: N512