Name: Techniques to Induce and Quantify Cellular Senescence (Video) | JoVE Journal
Uploaded: 2017-05-01T11:30:00.000+00:00
Duration: 6 min 51 s
Description: Learn techniques to induce and quantify cellular senescence using DNA damage and beta-galactosidase staining assays. These methods enable monitoring of senescence markers across diverse cell types for aging research.

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5 minutes

This study was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging. The authors wish to thank Myriam Gorospe and Kotb Abdelmohsen for many helpful discussions about senescence and Kotb Abdelmohsen for also critically reading the manuscript. We also thank our laboratory members, especially Douglas Dluzen for critically reading the manuscript.

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</ol>

The authors have nothing to disclose.

Here, we have described methods for replicative and DNA-damage induced senescence using human diploid fibroblasts. In addition, techniques for quantifying protein and mRNA levels of various senescence-associated proteins are included, as well as staining for SA-&#946;-gal and for the DNA-damage marker &#947;-H2AX. These protocols can be widely used to assess senescent phenotypes both in vitro and in vivo, although many caveats exist for characterizing senescence in vivo20</sup...

Over half a century ago, Hayflick and colleagues described how untransformed cells proliferate in culture, but for only a finite period of time1. Long-term culturing of human fibroblasts caused the cells to stop proliferating; however, they were metabolically active, and this was called cellular senescence. Senescence can be beneficial for inhibiting tumorigenesis, but it also can be detrimental, as it is thought to contribute to the loss of regenerative capacity that occurs with aging2,3. Senescent cells have been shown to accumulate in tissues as humans age4 and have been implicated in a number of biological processes, including embryonic development, wound healing, tissue repair, and age-related inflammation2.
Continual passaging of cells in culture induces replicative senescence, which has been linked to telomere attrition and genomic instability. Various cell stresses, including DNA damage and oncogenes, can also cause senescence3. Senescence caused by factors other than telomere attrition is often called stress-induced or premature senescence and generally depends on the p16INK4A/Rb pathway5. Although proliferating, untransformed cells typically appear spindle in shape, senescent cells can be identified as having certain characteristics, including a flat, large morphology and increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal) (Figures 1 &#38; 2). Senescent cells also accumulate DNA damage markers, including &#947;-H2AX (Figure 3)6, and, potentially, senescence-associated heterochromatin foci (SAHF) (Figure 4)7. Senescent cells have higher levels of cell cycle regulators, including p16 (p16INK4A) and/or p21 and p53 (Figure 5)8,9. Moreover, recent data have shown that senescent cells can have non-autonomous effects by secreting a number of pro-inflammatory cytokines and chemokines called the senescence-associated secretory phenotype (SASP)10. Although this SASP phenomenon may vary from cell type to cell type, in general, it is demonstrated by an increase in Interleukin-6 (IL-6), IL-8, granulocyte-macrophage colony stimulating factor (GM-CSF), growth-regulated oncogene &#945; (GRO-&#945;), and GRO-&#946;, among others (Figure 6). The particular stress or damage that induces senescence may also influence the secretory phenotype11,12,13. SASP can be detected by measuring the levels of secreted proteins using ELISAs or cytokine/protein arrays10,14. Although post-transcriptional mechanisms can regulate SASP protein levels11,15,16,17, changes in mRNA levels can also be detected in many cases. These changes are generally more sensitive and easier to quantify than protein level measurements. Other senescent markers can also be assessed, including persistent DNA damage nuclear foci, called DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)18, and various other markers3,19,20.
Here, we describe common techniques for inducing senescence in cells in culture and also for measuring several markers of senescence, including SA-&#946;-Gal, &#947;-H2AX, SAHF, and the protein and mRNA of senescence-associated molecules.

<table><tbody><tr><td>16% Tris-glycine gels</td><td>Invitrogen</td><td>XP00160BOX</td><td></td></tr><tr><td>Acid-Phenol ChCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Ambion</td><td>AM9720</td><td></td></tr><tr><td>Alexa-Fluor 568 goat anti-mouse antibody</td><td>Invitrogen</td><td>A11031</td><td>1:300 dilution</td></tr><tr><td>Cell lifters</td><td>Corning Inc.</td><td>3008</td><td>Cell scraper</td></tr><tr><td>ECL anti-mouse HRP linked antibody</td><td>Amersham</td><td>NA931V</td><td></td></tr><tr><td>ECL Plus Western Blotting Substrate</td><td>Pierce</td><td>32132</td><td>ECL</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>MP01306</td><td>stock 5 mg/mL in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>GAPDH antibody</td><td>Santa Cruz</td><td>sc-32233</td><td>1:1,000 - 5,000 dilution</td></tr><tr><td>GlycoBlue</td><td>Ambion</td><td>AM9515</td><td></td></tr><tr><td>Histone H3 dimethyl K9 monoclonal antibody</td><td>Abcam</td><td>1220</td><td>1:500 dilution</td></tr><tr><td>Human IL-6 Quantikine ELISA assay</td><td>R&amp;amp;D systems</td><td>D6050</td><td></td></tr><tr><td>Human IL-8 Quantikine ELISA assay</td><td>R&amp;amp;D systems</td><td>D8000C</td><td></td></tr><tr><td>Human GROa Quantikine ELISA assay</td><td>R&amp;amp;D systems</td><td>DRG00</td><td></td></tr><tr><td>N-N-dimethylformamide&amp;nbsp;</td><td>Sigma</td><td>D4551</td><td>DMF</td></tr><tr><td>p16 monoclonal antibody</td><td>BD Biosciences</td><td>51-1325gr</td><td>1:500 dilution</td></tr><tr><td>p21 monoclonal antibody</td><td>Millipore</td><td>05-345</td><td>1:750 dilution</td></tr><tr><td>p53 monoclonal antibody</td><td>Santa Cruz</td><td>sc-126</td><td>1:500 dilution clone DO-1</td></tr><tr><td>phospho-H2AX (Ser139) FITC conjugate antibody</td><td>Cell Signaling</td><td>9719</td><td>1:2,000 dilution</td></tr><tr><td>&lt;em&gt;POWER&lt;/em&gt; SYBR-green PCR master mix&amp;nbsp;</td><td>Applied Biosystems</td><td>4367659</td><td></td></tr><tr><td>Pre-stained molecular weight markers</td><td>Biorad</td><td>161-0374</td><td></td></tr><tr><td>ProLong Gold Antifade&amp;nbsp;</td><td>Invitrogen</td><td>P36930</td><td></td></tr><tr><td>PVDF membrane&amp;nbsp;</td><td>Thermo Scientific</td><td>88518</td><td></td></tr><tr><td>Senescence &amp;beta;-Galactosidase Staining Kit</td><td>Cell Signaling</td><td>9860</td><td></td></tr><tr><td>TRIzol</td><td>Ambion/Life Tech</td><td>10296028</td><td></td></tr></tbody></table>

techniques to induce and quantify cellular senescence

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Figures 2 - 6 show representative results from SA-β-gal staining; staining for γ-H2AX and SAHF; assessment of protein levels of p16INK4A, p21, and p53; and mRNA and protein levels of senescent-associated molecules. Increased SA-β-gal staining occurs with replicative and DNA damage-induced senescence. Also, observe the morphological changes that occur with senescence. Cells become enlarged and flat compared to the spindle appearance of proliferating fibroblasts....

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

The overall goal of these techniques is to provide a toolbox for enabling the induction, monitoring, and quantification of cellular senescence. These methods can help answer key questions about the identification and quantification of cellular senescence and the biologic factors that regulate cellular and organismal aging. The main advantage of these techniques is that they encompass a variety of methods for monitoring cellular senescence in different models, tissues, and cell types.Begin by plating the early passage proliferative cells of interest at a sparse density in the appropriate dish for the analysis. And culture the cells overnight in a tissue culture incubator. When the cells are 50-60%confluent, aspirate the growth medium and wash the cells with PBS two times.After the second wash, cover the cells with a thin layer of PBS. And expose the cells to a single 10 grade dose of gamma irradiation. Replace the PBS with growth medium.And return the cells to the incubator. Changing the medium every 48 to 72 hours with daily monitoring for morphological changes under a light microscope. Seven to 10 days after the irradiation, thaw all of the reagents from a commercial senescence associated beta-galactosidase assay kit in a 37 degree Celsius water bath.When all of the reagents reach room temperature, prepare the fixative and staining solutions according to the manufacturer's instructions. And gently wash each well with room temperature PBS. Take care to rinse the cells carefully as senescence cells do not adhere well and can be easily removed during washes.Fix the cells in each well with one milliliter of fixative solution per well for 10 to 15 minutes at room temperature. Followed by two two milliliter washes with PBS per well. Next, add one milliliter of beta-galactosidase staining solution to each well and cover the plate with aluminum foil for a 24 to 48 hour culture in a 37 degrees Celsius incubator without carbon dioxide.Check the cells at 24 hours. When the cells are sufficiently stained, replace the beta-gal solution with fresh PBS and examine the cultures on a light microscope with an attached camera at a 10x or a higher objective. The senescence associated beta-galactosidase cells will appear blue.Then, obtain at least five non-overlapping images per well. And manually count the total number of senescence associated beta-galactosidase positive cells and the total number of cells per image to calculate the percentage of senescence associated beta-galactosidase cells per well. 10 days after gamma irradiation, wash the cells three times with PBS.After the last wash, replace the PBS with 2.5 milliliters of serum free medium, supplemented with antibiotics per six centimeter dish and return the cells to the tissue culture incubator overnight. The next morning, transfer the medium into a conical tube on ice. And wash the cells two times with fresh PBS.Then, trypsinise and count the cells. Next, centrifuge the supernatant harvested from the cell culture plate. And then filter it through a 0.45 micrometer syringe strainer and freeze the medium in 500 microliter aliquots at 80 degree Celsius until their analysis by ELISA.Increased senescence associated beta-gal staining occurs with replicative and DNA damage induced senescence. Note the enlarged flat shape of the senescent cells compared to the spindle-like appearance of the proliferating fibroblasts. In this experiment, proliferating young or replicative senescent cells were fixed as described in the written protocol.And stained with an anti-body against a bio marker for DNA double strand breaks and DAPI. With an apparent increase in the number of DNA double strand break fosi observed in the senescent cells. RT-qPCR analysis of senescence associated secretory phenotype factors reveals that at least some factors are up regulated after gamma irradiation induced senescence in low passage human diploid fibroblasts at the MRNA level.Which is further confirmed by ELISA of the gamma irradiated cell culture supernatants. Once mastered, the DNA damage induction and SA beta-gal staining can be completed in approximately 30 minutes of hands-on time. The condition media for quantifying SASP protein levels can be prepared in about 30 minutes.While attempting this procedure, it's important to remember to monitor them undergoing morphological changes in the cell cultures before proceeding to the senescent marker quantification. After its development, these techniques paved the way for researchers in the fields of aging and cancer to explore senescence in a variety of tissues, cell cultures, and model organisms. After watching this video, you should have a good understanding of how to induce, monitor, and quantify cellular senescence.

techniques-to-induce-and-quantify-cellular-senescence

Research

JoVE Journal

Biology

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used β-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

A Sensitive Method to Quantify Senescent Cancer Cells

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Induction and Validation of Cellular Senescence in Primary Human Cells

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells.

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Multiplexed immunofluorescence enables sequential labeling of multiple antibody markers within a single tissue section to support spatial analysis of tissue architecture and cell populations. Here, a workflow for multiplex detection of senescence-associated, structural, and immune markers is presented, including manual and automated slide preparation, iterative imaging, and single-cell analysis using digital pathology software.

Multiplexed Immunofluorescence Assay for Spatial Assessment of Senescence Markers in vivo

This article presents a live-cell LysoTracker protocol to measure lysosomal remodeling during senescence in IMR-90 human fibroblasts.

Lysosomal Profiling With LysoTracker For Quantitative Assessment of Cellular Senescence In Human Fibroblasts

This method studies how senescent skin fibroblasts affect healthy ones. Senescence is induced via UVB exposure, and the conditioned medium is applied to normal fibroblasts. It helps evaluate bioactive compounds targeting altered cell-to-cell communication in skin aging.

Novel Combination Of Antioxidants To Restore Healthy Intercellular Communication In An In vitro Model Using Conditioned Medium From Senescent Cells

Learn techniques to induce and quantify cellular senescence using DNA damage and beta-galactosidase staining assays. These methods enable monitoring of senescence markers across diverse cell types for aging research.

Techniques to Induce and Quantify Cellular Senescence

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