A Practical Guide to Assessing Cellular Senescence

The biological phenomenon of cellular senescence brings about permanent cell cycle arrest when cells encounter stressors such as DNA damage, telomere shortening, or oncogene activation. Because scientists increasingly acknowledge cellular senescence as essential to aging and related diseases, research requires dependable biomarkers for its measurement. The biomarkers for cellular senescence are separated into primary and secondary types, which stand for unique features of the senescence phenotype. The manual presents a comprehensive workflow for cellular senescence assessment and outlines methods to identify both primary and secondary biomarkers.

Fig. 1 General hallmarks of cellular senescence. (González-Gualda E, et al., 2021)

Biomarkers of Cellular Senescence

Senescent cells maintain stable cell cycle arrest along with changes in morphology and metabolism while exhibiting chromatin restructuring and altered gene expression patterns as they develop the senescence-associated secretory phenotype (SASP). The presence of all biomarkers linked to senescence is not guaranteed in senescent cells. Some biomarkers used to detect senescent cells also appear in apoptotic cells and quiescent cells, making them unreliable for the exclusive identification of senescence. The detection of senescent cells demands the presence of several specific biomarkers.

Cell Cycle Arrest

Cellular senescence manifests primarily as a permanent stoppage of cell cycle progression during the G1 phase. Specific inhibitors of cyclin-dependent kinases, including p16^INK4a and p21^CIP1, control the mechanism for cell cycle arrest. The heightened activity of these particular factors inhibits cell division, thus preventing damaged cells from replicating.

Morphological Alterations

Senescent cells undergo specific morphological changes characterized by enlarged cell size, called hypertrophy, together with flattened cell shape and modifications in nuclear structure. Cytoskeletal reorganization combined with changes in gene expression patterns create these modifications.

Metabolic Changes

Senescent cells undergo metabolic shifts that include both increased glycolytic activity and transformation of mitochondrial function. Shifts between metabolic pathways and the generation of reactive oxygen species (ROS) usually occur together.

DNA Damage and DNA Damage Response (DDR)

Cellular senescence starts when DNA damage occurs. Stress factors result in DNA damage in cells, which subsequently triggers the activation of the DDR pathway. The DDR pathway executes its function using key proteins p53, ATM, and ATR, which identify damage and manage senescence processes.

Workflow for Assessing Cellular Senescence

The assessment of cellular senescence includes a systematic process starting with senescence induction in cells and allowing sufficient time for the senescence to develop before conducting a thorough examination of the senescent phenotype's defining traits.

Fig. 2 Combinatorial approaches to detect and assess cellular senescence. (González-Gualda E, et al., 2021)

• Induction of Senescence: Cells need defined periods to enter full senescence through chemotherapy or irradiation over 7-10 days, while oncogene overexpression requires 3-7 days.
• Assessment of Primary Characteristics of Senescence: The primary characteristic analysis of senescence starts with testing cell cycle arrest via cell cycle markers and confirming the end of cellular proliferation. The subsequent stage involves checking structural alterations associated with senescence by examining lysosomal mass increase (SA-β-gal) and organelle structure changes (Lamin B1 downregulation) while assessing epigenetic markers (SAHFs).
• Verification of Senescent Phenotype: A senescence-specific marker, such as DDR markers or increased ROS levels or SASP expression, confirms the senescent phenotype, which completes the process.

Detection of Primary Senescence Markers

Detection of Cell Cycle Arrest

• Evaluation of Key Protein Markers: Two primary pathways that involve the p16Ink4a/RB and p53/p21CIP1 axes mediate cell cycle arrest in senescent cells. To evaluate cell cycle arrest in senescent cells, researchers must assess multiple protein markers such as p16, p21, and p53, along with reduced phosphorylation of Retinoblastoma protein (pRB).
• Cellular Proliferation and DNA Replication Assays: The evaluation of cell cycle arrest is performed by researchers through simultaneous measurement of cellular proliferation levels and DNA replication tests. A combination of live automated cell monitoring along with spectrophotometric measurements at set time intervals enables researchers to generate and analyze in vitro cellular growth curves. The process of DNA synthesis measurement involves following the incorporation of modified nucleotides into the DNA of replicating cells.
• Quantification of mRNA Levels: Target mRNAs can be measured through the process of quantitative real-time PCR (qPCR). Current research reveals that housekeeping genes traditionally used for normalization exhibit altered expression patterns during cellular senescence.

Detection of Senescence-Associated β-Galactosidase (SA-β-Gal) Activity

• SA-β-Gal Staining: Using the specific substrate X-gal incubation causes cells to form a blue precipitate when SA-β-Gal is active. Both microscopy and flow cytometry can detect this stain.
• Lysosomal Activity Assays: Higher lysosomal quantities produce amplified fluorescence signals when researchers use LysoTracker dyes to stain acidic organelles.

Detection of Nuclear Alterations

• Fluorescence Microscopy: Cells can be stained with DNA-binding dyes such as DAPI, which enables researchers to visualize nuclear morphology. Through microscopy, scientists can detect differences in cell size and shape.
• Immunofluorescence: The use of senescence-associated heterochromatin markers, including γH2AX, allows scientists to identify DNA damage and chromatin changes through specialized staining methods.

Detection of Secondary Senescence Markers

Detection of Cytokine Secretion

• Cytokine Arrays: Multiplex assays measure exact cytokine levels quantitatively in culture media. Separate cytokine molecules can be studied by researchers using purchased ELISA kits.
• RNA Analysis: The combination of RNA sequencing and qPCR enables scientists to quantify gene expression profiles that are associated with senescence, including those genes that make up the SASP.

Detection of Mitochondrial Changes

Specific fluorescent dyes help researchers evaluate mitochondrial mass by staining active mitochondria. Scientists use either flow cytometry or fluorescence microscopy to measure fluorescence intensity. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) function as dual indicators for mitochondrial health and metabolic shifts.

Detection of Reactive Oxygen Species

• Fluorescent Probes: The fluorescent dye DCFDA (2',7'-dichlorofluorescin diacetate) allows researchers to quantify the levels of ROS. DCFDA transforms into a fluorescent molecule upon reacting with ROS, which allows researchers to detect ROS levels by using flow cytometry or fluorescence microscopy.
• Electron Paramagnetic Resonance (EPR): This technique allows scientists to measure ROS concentrations directly inside cells and tissues while providing exact counts of specific ROS species.

Detection of Morphology and Granularity of Senescent Cells

• Microscopy: Both light microscopy and fluorescence microscopy prove useful when it comes to identifying changes in cell morphology. DAPI staining provides detailed visualization of nuclear dimensions and structure, whereas other staining procedures reveal cytoplasmic characteristics.
• Flow Cytometry: Scientists utilize this technique to conduct assessments of cellular dimensions as well as granularity levels. The forward scatter (FSC) measurement determines cell size, while side scatter (SSC) evaluates cell granularity and internal structure. A rise in SSC levels indicates that senescent cells show alterations in their granularity.
• Quantitative Image Analysis: Scientists can assess quantitative morphological changes in senescent cells with advanced imaging software that evaluates parameters related to cell area, perimeter, and circularity.

Understanding cellular senescence is crucial for revealing its effects on aging processes and disease development. CD BioSciences implements a multi-dimensional technique that focuses on identifying primary and secondary biomarkers to help clients understand senescence mechanisms and their consequences. If you are interested in our services, please feel free to contact us or make an online inquiry.

References

1. González-Gualda E, et al. A guide to assessing cellular senescence in vitro and in vivo. FEBS J, 2021, 288 (1): 56-80.
2. Bernardes de Jesus B, Blasco MA. Assessing cell and organ senescence biomarkers. Circ Res, 2012, 111 (1): 97-109.