Name: techniques to induce and quantify cellular senescence
Uploaded: 2017-05-01T11:30:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about Techniques to Induce and Quantify Cellular Senescence at JoVE.com

这项研究是由美国国立卫生研究院老龄研究所院内研究计划的支持。作者要感谢迈娅姆·戈罗斯佩和寇柏Abdelmohsen约衰老和寇柏Abdelmohsen的批判也读手稿许多有益的讨论。我们也感谢我们的实验室成员，特别是道格拉斯·德卢岑批判地阅读手稿。

作者什么都没有透露。

在这里，我们已经描述了使用人二倍体成纤维细胞用于复制和DNA损伤诱导的衰老的方法。此外，用于定量蛋白质和多种衰老相关蛋白的mRNA水平的技术包括，以及染色SA-β-gal和对DNA损伤标记物γ-H2AX。这些协议可广泛用于评估在体外和体内表型衰老，虽然用于体内 20表征衰老存在许多警告。其他细胞可表达衰老标记，即使它们不衰老细胞。例如，细胞如破骨细?...

半个多世纪前，海弗利克和他的同事描述了细胞是如何在未转化增殖的文化，但只是时间1有限的时期。人成纤维细胞的长期培养导致细胞增殖停止;然而，他们是代谢活跃，这被称为细胞衰老。衰老可用于抑制肿瘤发生是有利的，但它也可能是有害的，因为它被认为有助于其与衰老2,3发生的再生能力的损失。衰老细胞已被证明在组织中积累作为人类4年龄和在许多生物过程，包括胚胎发育，伤口愈合，组织修复，和年龄相关的炎症2的有牵连。 在培养细胞的持续传代引起复制性衰老，这已链接以端粒耗损和基因组不稳定性。各种细胞的应力，包括DNA损伤和癌基因，也可引起衰老3。由除端粒耗损等因素衰老经常被称为应力诱导或早衰和通常取决于P16 INK4A / Rb途径5。虽然增殖，未转化的细胞通常出现主轴在形状，衰老细胞可被识别为具有特定的特征，包括一平的，大的形态和提高衰老相关β半乳糖苷酶的活性（SA-β-GAL）（ 图1和2）。衰老细胞也积累的DNA损伤标记物，包括γ-H2AX（ 图3）6，以及潜在的衰老相关异灶（SAHF）（ 图4）7。衰老细胞中具有较高水平的细胞周期调节的，包含p<html> 16（P16 INK4A）和/或p21蛋白和p53（ 图5）8,9。此外，最近的数据已经表明，衰老细胞可通过分泌许多促炎性细胞因子和趋化因子具有非自主效应称为衰老相关分泌表型（SASP）10。虽然这种现象SASP可以从细胞类型而有所不同，以细胞类型，一般地，它是通过增加白细胞介素-6（IL-6），IL-8，粒细胞 - 巨噬细胞集落刺激因子（GM-CSF），生长 - 证明调节的原癌基因α（GRO-α），和GRO-β，等等（ 图6）。诱导衰老的特定应力或损坏还可能影响分泌表型11，12，13。 SASP可以通过测量使用的ELISA或细胞因子/蛋白质阵列分泌的蛋白质的水平来检测S = "外部参照"&gt; 10，14。尽管转录后的机制可以调节SASP蛋白水平11，15，16，17，在mRNA水平的变化也可以在许多情况下检测。这些变化通常更敏感，更容易比蛋白质水平的测量来量化。其它衰老标记物也可以被评估，包括持久性DNA损伤核灶，与染色质的改变称为DNA片段增强衰老（DNA-SCARS）18，以及各种其它的标记3，19，20。 在这里，我们将介绍常见的技术在培养细胞诱导的衰老，也为测量衰老的几个标志物，包括SA-β-Gal的，γ-H2AX，SAHF和衰老的蛋白和mRNANCE相关分子。

<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

响应于细胞应激或损害，增殖细胞可诱导特定的程序发起的长期细胞周期停滞的状态，称为细胞衰老。衰老细胞积累的有机体衰老和通过体外培养持续发生。衰老细胞中影响许多生物过程，包括胚胎发育，组织修复和再生，肿瘤抑制和老化。衰老细胞的标志包括，但不限于，增加的衰老相关β半乳糖苷酶的活性（SA-β-GAL）; P16 INK4A和p53和p21的水平;更高水平的DNA损伤，包括γ-H2AX;衰老相关异染色质灶（SAHF）的形成;和获取一个衰老相关分泌表型（SASP），这种现象的特征在于数促炎细胞因子和信号分子的分泌的。在这里，我们描述协议都复制和DNA损伤诱导的衰老中培养的细胞。另外，我们强调技术使用几种衰老相关的标志物，包括SA-β-GAL，γ-H2AX和SAHF染色监测衰老表型，并量化细胞周期调控和SASP因子蛋白和mRNA水平。这些方法可以适用于衰老的各种型号和组织的评估。

图2 -图6示出从SA-β-gal染色的代表性结果;染色γ-H2AX和SAHF; P16 INK4A，P21和p53的蛋白水平的评估;和mRNA和衰老相关分子的蛋白水平。增加SA-β-gal染色以复制和DNA损伤诱导的衰老发生。此外，观察与衰老发生形态变化。相比增殖的成纤维细胞的主轴外观细胞成为扩大且平坦。对于此实验范例，细胞染色和RNA /蛋白质被暴露于IR辐射之后，分离7天。更健壮的结?...

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

Techniques to Induce and Quantify Cellular Senescence

细胞衰老，细胞周期停滞的不可逆状态，可通过各种细胞应激来诱导。在这里，我们描述的协议来诱导细胞衰老和方法来评估衰老的标志。

技术诱导和量化细胞衰老

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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SCIENTISTS IN ACTION

33.1K 次观看.  National Institute on Aging, National Institutes of Health. 细胞衰老，细胞周期停滞的不可逆状态，可通过各种细胞应激来诱导。在这里，我们描述的协议来诱导细胞衰老和方法来评估衰老的标志。 

视频: 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.

微机电与细胞生物学研究所：展望与应用

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

科研

生物学

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.

骨骼肌损伤诱导衰老和体内重编程的评价

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

发育生物学

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.

量化衰老癌症细胞的敏感方法

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

医学

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.

原发性细胞衰老的诱导与鉴定

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.

基因型衰老过程中正常人成纤维细胞活性氧和衰老相关分泌表型的定量测定

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.

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

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

癌症研究

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.

远红荧光衰老相关β-半乳糖苷酶探针，用于通过流式细胞术鉴定和富集衰老肿瘤细胞

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.

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

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

生物化学

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.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

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.

基于流式细胞术的治疗诱导的衰老癌细胞定量

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