Questo studio è stato sostenuto dal programma di ricerca intramurale del National Institutes of Health, National Institute on Aging. Gli autori desiderano ringraziare Myriam Gorospe e Kotb Abdelmohsen per molte discussioni utili su senescenza e Kotb Abdelmohsen per anche criticamente la lettura del manoscritto. Ringraziamo anche i nostri membri di laboratorio, in particolare Douglas Dluzen per la lettura critica del manoscritto.

Gli autori non hanno nulla da rivelare.

Qui, abbiamo descritto metodi per replicativo e danno del DNA senescenza indotta utilizzando fibroblasti diploidi umane. Inoltre, le tecniche di quantificazione delle proteine ​​e livelli di mRNA di varie proteine ​​senescenza associate sono inclusi, così come colorazione per SA-β-gal e per il marcatore DNA-danneggiamento γ-H2AX. Questi protocolli possono essere ampiamente utilizzati per valutare fenotipi senescenti sia in vitro che in vivo, sebbene esistano molti avvertimenti per la caratte...

Più di mezzo secolo fa, Hayflick e colleghi hanno descritto come le cellule proliferano non trasformata in cultura, ma solo per un periodo limitato di tempo 1. coltura a lungo termine di fibroblasti umani ha causato le cellule per fermare la proliferazione; tuttavia, erano metabolicamente attiva, e questo è stato chiamato senescenza cellulare. Senescenza può essere utile per inibire la tumorigenesi, ma può anche essere dannoso, in quanto si ritiene contribuiscano alla perdita di capacità rigenerativa che si verifica con l&#39;invecchiamento 2, 3. Cellule senescenti hanno dimostrato di accumularsi nei tessuti come esseri umani 4 anni e sono stati implicati in una serie di processi biologici, tra cui lo sviluppo embrionale, la guarigione delle ferite, la riparazione dei tessuti e l&#39;infiammazione legata all&#39;età 2. passaging continuo di cellule in coltura induce senescenza replicativa, che è stato collegatodi telomero attrito e instabilità genomica. Varie sollecitazioni cellulari, comprese danno al DNA e oncogeni, possono anche causare senescenza 3. Senescenza causata da fattori diversi telomere attrito è spesso chiamato da stress o senescenza prematura e dipende generalmente dalla p16 INK4A / Rb pathway 5. Sebbene proliferanti, cellule non trasformate compaiono tipicamente mandrino in forma, cellule senescenti possono essere identificati come aventi determinate caratteristiche, tra cui una TV, ampio morfologia e una maggiore attività β-galattosidasi senescenza-associato (SA-β-gal) (Figure 1 e 2). Cellule senescenti accumulano anche marcatori di danno del DNA, tra cui γ-H2AX (figura 3) 6, e, potenzialmente, senescenza associata eterocromatina foci (SAHF) (Figura 4) 7. cellule senescenti hanno livelli elevati di regolatori del ciclo cellulare, tra cui p<html> 16 (INK4A p16) e / o p21 e p53 (Figura 5) 8, 9. Inoltre, i dati recenti hanno dimostrato che le cellule senescenti possono avere effetti non autonomi secernendo un numero di citochine pro-infiammatorie e chemochine chiamato senescenza associata secretoria fenotipo (PSAS) 10. Sebbene questo fenomeno SASP può variare da tipo cellulare a tipo cellulare, in generale, è dimostrato da un aumento di interleuchina-6 (IL-6), IL-8, fattore stimolante le colonie di granulociti-macrofagi (GM-CSF), alla crescita α regolata oncogene (GRO-α) e GRO-β, tra gli altri (Figura 6). Lo stress particolare o danni che induce senescenza possono anche influenzare il fenotipo secretorio 11, 12, 13. SASP può essere rilevata misurando i livelli di proteine ​​secrete mediante ELISA o array citochine / proteinas = &quot;xref&quot;&gt; 10, 14. Sebbene i meccanismi post-trascrizionali possono regolare i livelli proteici SASP 11, 15, 16, 17, cambiamenti nei livelli di mRNA possono essere rilevati anche in molti casi. Questi cambiamenti sono generalmente più sensibili e più facile da quantificare rispetto misurazioni del livello di proteine. Altri marcatori senescenti possono anche essere valutati, compresi persistente danno al DNA foci nucleari, detti segmenti di DNA con alterazioni della cromatina rinforzo senescenza (DNA-SCARS) 18, e vari altri marcatori 3, 19, 20. Qui, descriviamo comuni tecniche per indurre senescenza in cellule in coltura e per la misura diversi marcatori di senescenza, tra SA-β-Gal, γ-H2AX, SAHF, e la proteina e mRNA di senescemolecole SNO-associata.

<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

In risposta allo stress cellulare o danno, le cellule proliferanti possono indurre un programma specifico che avvia uno stato di arresto del ciclo cellulare a lungo termine, chiamato senescenza cellulare. Accumulo di cellule senescenti verifica con l&#39;invecchiamento organismal e mediante coltura continua in vitro. cellule senescenti influenzano molti processi biologici, compreso lo sviluppo embrionale, la riparazione dei tessuti e la rigenerazione, soppressione del tumore, e l&#39;invecchiamento. Caratteristiche di cellule senescenti includono, ma non sono limitati a, maggiore attività β-galattosidasi senescenza-associato (SA-β-gal); p16 INK4a, p53, p21 e livelli; Livelli più elevati di danno al DNA, tra γ-H2AX; la formazione di senescenza associata Eterocromatina Foci (SAHF); e l&#39;acquisizione di un senescenza associata secretoria fenotipo (SASP), un fenomeno caratterizzato dalla secrezione di alcune citochine pro-infiammatorie e molecole di segnalazione. Qui, descriviamo i protocolli sia per replicativo esenescenza DNA danno-indotta in cellule coltivate. Inoltre, si segnalano tecniche per monitorare il fenotipo senescente utilizzando diversi marcatori senescenza-associata, tra SA-β-gal, γ-H2AX e SAHF colorazione, e per quantificare i livelli di proteine ​​e mRNA di regolatori del ciclo cellulare e fattori SASP. Questi metodi possono essere applicati alla valutazione della senescenza in vari modelli e tessuti.

Le figure 2 - 6 mostrano risultati rappresentativi SA-β-gal colorazione; colorazione per γ-H2AX e SAHF; valutazione dei livelli di proteina di p16 INK4A, p21, e p53; e mRNA e di proteina di molecole senescenti-associata. Aumento della colorazione SA-β-gal verifica con replicativa e danni al DNA indotto senescenza. Inoltre, osservare i cambiamenti morfologici che si verificano con senescenza. Cellule diventano allargata e piatta rispetto alla comparsa del ma...

Watch this Scientific Journal Video about Techniques to Induce and Quantify Cellular Senescence at JoVE.com

Techniques to Induce and Quantify Cellular Senescence

senescenza cellulare, lo stato irreversibile di arresto del ciclo cellulare, può essere indotta da vari stress cellulari. Qui, descriviamo i protocolli per indurre la senescenza e metodi per valutare cellulare marcatori di senescenza.

Le tecniche per indurre e Quantificare cellulare senescenza

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 ...

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

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33.0K Views.  National Institute on Aging, National Institutes of Health.  senescenza cellulare, lo stato irreversibile di arresto del ciclo cellulare, può essere indotta da vari stress cellulari. Qui, descriviamo i protocolli per indurre la senescenza e metodi per valutare cellulare marcatori di senescenza. 

Video: Techniques to Induce and Quantify Cellular Senescence

BioMEMS and Cellular Biology: Perspectives and Applications

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in drug screening, toxicology, and clinical assays, and is increasingly used in regenerative medicine. However, the traditional cell culture methodology   essentially consisting of the immersion of a large population of cells in a homogeneous fluid medium and on a homogeneous flat substrate   has become increasingly limiting both from a fundamental and practical perspective. Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, and the medium composition, as well as the neighboring cell type in the surrounding cellular microenvironment. Additionally, microtechnology is conceptually well-suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. In this interview, Albert Folch explains these limitations, how they can be overcome with soft lithography and microfluidics, and describes some relevant examples of research in his lab and future directions.

BioMEMS e Biologia Cellulare: prospettive e applicazioni

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in ...

Ricerca

Biologia

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

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

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

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.

Valutazione delle lesioni-indotta senescenza e In Vivo riprogrammazione nel muscolo scheletrico

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Biologia dello sviluppo

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

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 &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Un metodo sensibile per quantificare le cellule tumorali senescenti

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Medicina

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

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.

Induzione e convalida della senescenza cellulare in cellule umane primarie

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

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

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

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.

Una misura quantitativa di specie reattive dell'ossigeno e senescenza-collegata di fenotipo secretivo in fibroblasti umani normali durante la senescenza indotta da Oncogene

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

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

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

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

Imaging simultaneo e rilevamento basato sulla citometria a flusso di marcatori di senescenza fluorescenti multipli in cellule tumorali senescenti indotte dalla terapia

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Ricerca sul cancro

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

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

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.

Sonda β-galattosidasi associata alla senescenza fluorescente Far-Red per l'identificazione e l'arricchimento delle cellule tumorali senescenti mediante citometria a flusso

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

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

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

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.

Misurazione dei tassi di turnover proteico in cellule coltivate senescenti e non in divisione con etichettatura metabolica e spettrometria di massa

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

Biochimica

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

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

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 &#946;-Galactosidase activity in single cells.

Saggio di screening basato su SA-Galactosidase per l'identificazione dei farmaci senoterapici

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells

Source: Flor, A., et al. <a href="https://app.jove.com/v/64176">Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry.</a> J. Vis. Exp. (2022)
This video illustrates a flow cytometry-based assay for quantifying therapy-induced senescent cancer cells with dual senescence markers. The detection and quantification of senescent cells involve using a &#946;-galactosidase substrate. The substrate is cleaved by active lysosomal &#946;-galactosidase within the senescent cells, producing a fluorescent product that, in conjunction with autofluorescent lipofuscin granules, marks the senescent cancer cells.

Quantificazione basata sulla citometria a flusso di cellule tumorali senescenti indotte da terapia

1. Induction of senescence by chemotherapy drugs in cultured cancer cells
NOTE: All cell manipulation steps in this section should be performed in a ...