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

Bu çalışma Ulusal Sağlık Enstitüleri İntramural Araştırma Programı, Ulusal Yaşlanma Enstitüsü tarafından desteklenmiştir. Yazarlar ayrıca eleştirel el yazması okumak için yaşlanma ve kotb Abdelmohsen ilgili birçok yararlı tartışmalar için Myriam Gorospe ve kotb Abdelmohsen teşekkür etmek istiyorum. Biz de eleştirel el yazması okumak için, özellikle Douglas Dluzen bizim laboratuvar üyelerine teşekkür.

Yazarlar ifşa hiçbir şey yok.

Burada, insan diploid fibroblastlarının kullanılarak replikatif ve DNA hasarına neden olduğu yaşlanma için yöntemler tarif etmişlerdir. Buna ek olarak, protein ve çeşitli senesans ile ilişkili proteinlerin mRNA seviyelerini ölçmek için teknikler, aynı zamanda boyama SA-β-gal ve DNA hasarına işaret γ-H2AX için edilmektedir. Birçok nokta vivo 20 yaşlanmayı karakterize etmek için mevcut olmakla beraber bu protokoller yaygın in vitro ve in vivo...

Yarım Üzeri asır önce, Hayflick ve arkadaşları kültürde çoğalmaya nasıl uğramamış hücreleri anlatılan ama zamanla 1&#39;in yalnızca sınırlı bir süre için. İnsan fibroblast uzun süreli kültürleme çoğalan durdurmak için hücrelerin neden olduğu; ancak, metabolik olarak aktif olduğunu ve bu hücresel yaşlanma olarak adlandırıldı. Yaşlılık tümör oluşumunu inhibe etmek için yararlı olabilir, ama yaşlanma 2, 3 oluşur rejeneratif kapasitesinin kaybına katkıda bulunduğu düşünülmektedir olarak aynı zamanda, zararlı olabilir. Yaşlanmış hücreler Gı&#39;de durdukları yaş 4 insanların ve embriyonik gelişme, yara iyileşmesi, doku tamiri, ve yaşla ilgili enflamasyon 2 de dahil olmak üzere biyolojik süreçler, bir dizi implike edilmiştir gibi dokularda birikebilir gösterilmiştir. kültürdeki hücrelerin sürekli pasajı bağlanmıştır replikatif senesansın, indükleryıpratma ve genomik istikrarsızlık telomer. DNA hasarı ve onkogenler de dahil olmak üzere çeşitli hücre gerilimleri, aynı zamanda yaşlanmayı 3 neden olabilir. Telomer yıpratma dışındaki faktörlerden kaynaklanan Yaşlılık genellikle stres kaynaklı veya vaktinden evvel yaşlanmaya denilen ve genel olarak p16 INK4a / Rb yolu 5 bağlıdır. Çoğalan birlikte, transforme olmamış hücreler tipik olarak şekil mili görünür yaşlanmış hücreleri, bazı özelliklere sahip bir düz, büyük bir morfoloji ve artan senesans ile ilişkili β-galaktosidaz aktivitesi (SA-β-gal) (Şekil 1 ve 2) dahil olmak üzere tespit edilebilir. Yaşlanmış hücreler de γ-H2AX (Şekil 3), 6, ve muhtemelen, senesans ile ilişkili heterokromatin odakları (SAHF) (Şekil 4) 7 de dahil olmak üzere, DNA hasarı belirteçler birikir. Yaşlanmış hücreler p dahil olmak üzere hücre döngü düzenleyicileri ile ilgili daha yüksek seviyelerde sahip<html> 16 (p16 INK4a) ve / veya p21 ve p53 (Şekil 5) 8, 9. Ayrıca, son veriler yaşlanmış hücrelerin, pro-inflamatuar sitokin ve kemokin, bir dizi salgılayarak otonom olmayan etkileri olduğu senesans ile ilişkili salgı fenotipi (SASP) 10 olarak adlandırılan göstermiştir. Bu SASP olgu, hücre türünden hücre türüne göre değişebilir, ancak genel olarak, bu İnterlökin-6 (IL-6) aksine, IL-8, granülosit-makrofaj koloni uyarıcı faktör (GM-CSF), büyümeyi bir artış ile ortaya konmuştur düzenlenmiş onkogen α (GRO-α) ve GRO-β, diğerleri arasında (Şekil 6). Yaşlanmayı indükleyen, özellikle stres veya hasar da salgı fenotipi 11, 12, 13 etkileyebilir. SASP ELISA&#39;lar veya sitokin / protein dizileri kullanılarak salgılanan proteinlerin seviyelerini ölçerek tespit edilebilirs = &quot;xref&quot;&gt; 10, 14. Transkripsiyon sonrası mekanizmalar SASP protein seviyeleri 11, düzenler, ancak 15, 16, 17, mRNA seviyelerinde değişiklikler de birçok durumda tespit edilebilir. Bu değişiklikler genellikle daha duyarlı ve protein seviyesi ölçümlerine göre ölçmek daha kolaydır. Diğer yaşlanmış belirteçler kalıcı DNA hasarı nükleer odaklar, yaşlanmayı (DNA izler) 18 takviye edici kromatin değişiklikler ile adlandırılan DNA bölümlerinin, ve çeşitli diğer belirteçler 3, 19, 20 dahil olmak üzere, saptanabilir. Burada, SA-β-Gal, γ-H2AX, SAHF da dahil olmak üzere yaşlanma ait birçok belirteç, ölçülmesi için kültürdeki hücreler yaşlanmayı uyarılması ve ortak teknikleri tarif eder ve yaşlanmaya protein ve mRNAmoleküller nce ilişkili.

<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

Hücresel stres veya hasara yanıt olarak, çoğalan hücrelerin hücresel yaşlanma olarak adlandırılan uzun süreli hücre siklüsü hapsi için bir durum başlatır belirli bir programı, neden olabilir. Yaşlanmış hücrelerin birikimi organizma yaşlanma ile ve in vitro olarak sürekli kültürleme yoluyla ortaya çıkar. Yaşlanan hücreler embriyonik gelişim, doku onarımı ve rejenerasyon, tümör bastırma ve yaşlanma gibi birçok biyolojik süreçleri, etkiler. yaşlanmış hücrelerin ayırt edici arasında, ancak, artan senesans ile ilişkili β-galaktosidaz aktivitesi (SA-β-gal) ile sınırlı değildir; p16 INK4a, p53 ve p21 seviyeleri; γ-H2AX dahil olmak üzere DNA hasarının daha yüksek seviyelerinin; Senesans ile ilişkili Heterokromatin odakları (SAHF) oluşması; ve bir senesans ile ilişkili Salgı fenotip (SASP), pro-enflamatuvar sitokinler ve sinyal molekülleri bir dizi salgılanması ile karakterize edilen bir fenomen kazanılması. Burada, hem replikatif için protokolleri tanımlamak veDNA, kültürlü hücrelerde yaşlanmayı hasar kaynaklı. Buna ek olarak, SA-β-gal, γ-H2AX ve SAHF boyama dahil olmak üzere birçok senesans ile ilişkili belirteçleri kullanarak yaşlanmış bir fenotipi izlemek, ve hücre döngüsü düzenleyicileri ve SASP faktörleri, protein ve mRNA seviyeleri ölçmek için teknikler vurgulamaktadır. Bu yöntemler, çeşitli model ve dokularında yaşlanmayı değerlendirilmesi için uygulanabilir.

, 6 SA-β-gal ile boyama temsil edici sonuçlar göstermektedir - 2 Şekil γ-H2AX ve SAHF için boyama; p16 INK4a, p21 ve p53 protein seviyelerinin değerlendirilmesi; ve mRNA ve yaşlanmış ilişkili moleküllerin protein seviyeleri. Artan SA-β-gal boyaması replikatif ve DNA hasarı ile indüklenen yaşlanma ile ortaya çıkar. Ayrıca, yaşlanma ile ortaya morfolojik değişikliklerini izleyin. Hücreler, çoğalan fibroblastların mil g...

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

Techniques to Induce and Quantify Cellular Senescence

Hücresel yaşlanma, hücre döngüsü tutuklama tersinmez durum, çeşitli hücresel stresler tarafından indüklenebilir. Burada, yaşlanmanın işaretlerini değerlendirmek için hücre yaşlanmasının ve yöntemleri ikna etmek protokolleri açıklar.

Teknikler İndüklemek ve Ölçmek Hücresel Yaşlılık etmek

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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Cell Proliferation

SCIENTISTS IN ACTION

33.0K Görüntüleme.  National Institute on Aging, National Institutes of Health.  Hücresel yaşlanma, hücre döngüsü tutuklama tersinmez durum, çeşitli hücresel stresler tarafından indüklenebilir. Burada, yaşlanmanın işaretlerini değerlendirmek için hücre yaşlanmasının ve yöntemleri ikna etmek protokolleri açıklar. 

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 ve Hücresel Biyoloji: Perspektifler ve Uygulamalar

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

Biyoloji

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.

Yaralanma kaynaklı yaşlanma ve In Vivo kas iskelet yeniden programlama değerlendirilmesi

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

Gelişim Biyolojisi

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.

Senesent Kanser Hücreleri ölçmek için bir Hassas Yöntem

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

Tıp

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.

İndüksiyon ve birincil insan hücrelerinde hücresel yaşlanma doğrulama

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.

Reaktif oksijen türleri ve yaşlanma ilişkili salgı fenotip Normal insan fibroblast içinde bir nicel ölçüm onkogen kaynaklı yaşlanma sırasında

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.

Tedaviye Bağlı Yaşlanan Kanser Hücrelerinde Çoklu Floresan Yaşlanma Belirteçlerinin Eşzamanlı Görüntüleme ve Akış Sitometrisine Dayalı Tespiti

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

Kanser Araştırmaları

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.

Akış Sitometrisi ile Yaşlanan Tümör Hücrelerinin Tanımlanması ve Zenginleştirilmesi için Uzak Kırmızı Floresan Yaşlanma İlişkili β-Galaktosidaz Probu

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.

Metabolik Etiketleme ve Kütle Spektrometrisi ile Yaşlanan ve Bölünmeyen Kültürlü Hücrelerde Protein Devir Hızlarının Ölçülmesi

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

Biyokimya

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.

Senoterapötik Ilaçların tanımlanması için SA-β-Galactosidaz tabanlı tarama tahlili

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.

Tedaviye Bağlı Yaşlanan Kanser Hücrelerinin Akış Sitometrisine Dayalı Miktar Tayini

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