Cellular senescence, which is marked by a permanent proliferative arrest, happens in response to both endogenous and exogenous stresses, such as telomere dysfunction, oncogene activation, and ongoing DNA damage. It is one of the major hallmarks of aging.
Fig. 1 Cellular senescence drivers and phenotypes. (Di Micco R, et al., 2021)
With aging, there is an accumulation of senescent cells in the organism. CD BioSciences uses a multi-dimensional approach combining various techniques such as flow cytometry, immunofluorescence, proteomics, and genomics to comprehensively analyze cellular senescence in aging.
Characterization of Cellular Senescence
Senescence-associated beta-galactosidase (SA-β-gal) activity assay
SA-β-gal is a widely used biomarker for cellular senescence. We employ advanced staining techniques to detect and quantify SA-β-gal activity, providing valuable insights into the presence and extent of cellular senescence. Our method requires no special equipment or manipulation and is suitable for cellular senescence analysis in vitro cell culture and in vivo tissue sections.
Cell cycle analysis
Our team uses flow cytometry to analyze cell cycle distribution, DNA content, and cell cycle regulatory proteins, which enables us to identify senescent cells and comprehend cell cycle arrest dynamics.
Senescence-associated secretory phenotype (SASP) analysis
We employ advanced proteomic techniques to profile the SASP components. So, we can evaluate the production and secretion of pro-inflammatory cytokines, chemokines, growth factors, and matrix-remodeling enzymes associated with the SASP, shedding light on the senescent cell secretome and its role in senescence-related inflammation.
Telomere length analysis
We utilize quantitative fluorescence in situ hybridization (Q-FISH) and quantitative PCR (qPCR) to measure telomere length, helping our clients understand the impact of telomere shortening on cellular senescence and its contribution to the aging process.
Analyzing the Mechanisms of Cellular Senescence
Senescence-associated DNA damage response (DDR) analysis
We utilize immunofluorescence and immunoblotting techniques to examine DDR markers, encompassing phosphorylated histone H2AX (γH2AX), p53, and p21, to clarify DNA damage signaling pathways involved in cellular senescence. Moreover, we assess the expression levels and activation status of DDR proteins, comprised of ATM, ATR, DNA-PK, p53, p21, and p16INK4a through immunofluorescence assays and Western blotting.
Telomere dysfunction analysis
We assess the length of telomeres and their activity of telomerase, whilst analyzing telomere dysfunction-induced foci (TIFs) to investigate the effects of telomere attrition on cellular senescence.
Oncogene-induced senescence analysis
We examine the expression levels and activity of oncogenes such as RAS, RAF, and others through RT-PCR, Western blotting, and immunohistochemistry in cells undergoing oncogene-induced senescence. We also assess the activation status of key signaling pathways, including the RAS/RAF/MEK/ERK and PI3K/AKT pathways, using phospho-specific antibodies and kinase activity assays to understand their role in senescence induction.
Our Platform for Detecting Cellular Senescence Markers
CD BioSciences takes pride in its platform for detecting cellular senescence markers in aging. Some of the common assays we use are cell staining, Western blotting, PCR, and ELISA. Our platform combines comprehensive technologies and expert analysis to provide accurate and reliable results.
| Characterization of cellular senescence |
Detection Indicators |
Detection Methods |
| Cell cycle arrest |
P15, p16, p21, p53, pRb, Ki67, Brdu |
WB, IHC, IF |
| DNA damage |
p-γH2AX, 53BP, ATM, ATR |
WB, IHC, IF |
| Metabolic changes |
SA-β-Gal, α-fucosidase, Lipofuscin |
IF, ELISA, optical microscope |
| Morphological changes |
Larger, flatter cells |
SEM electron microscope, optical microscope |
| SASP |
IL-6, IL-8 |
WB, IHC, IF |
| Apoptosis blockade |
Caspases, Bcl-2, cleaved PARP |
WB, IHC, IF, TUNEL assay |
Through the utilization of our advanced platform for detecting cellular senescence in the context of aging, Creative Bioarray helps our clients gain access to a versatile and detailed analysis solution, facilitating extensive exploration of this intricate biological process. If you are interested in our services, please feel free to contact us or make an online inquiry.
Reference
- Di Micco R, et al. Cellular senescence in aging: from mechanisms to therapeutic opportunities. Nat Rev Mol Cell Biol. 2021, 22 (2): 75-95.
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