Name: induction and validation of cellular senescence in primary human cells
Uploaded: 2018-06-20T14:30:00.000+00:00
Duration: 8 min 18 s
Description: Watch this Scientific Journal Video about Induction and Validation of Cellular Senescence in Primary Human Cells at JoVE.com

우리는 유익한 토론에 대 한 관리자 실험실 및 데이터와 UV-유도 노화에 프로토콜을 공유 하기 위한 Thijmen 밴 Vliet의 회원을 감사 합니다.

<ol>
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여기 설명 하는 프로토콜은 인간의 기본 섬유 아 세포, 최적화 된 특히 BJ와 WI-38 셀. 일차 노화, 전리 방사선, 독 소 루비에 대 한 프로토콜을 성공적으로 다른 세포 유형 즉 신생아 melanocytes와 keratinocytes (iPSC 파생 cardiomyocytes)과 섬유 아 세포 (HCA2 및 IMR90)의 다른 종류에 적용 우리의 실험실. 그러나, 몇 가지 세부 사항을 시드 셀, 방법 및 플라스틱 지원에 연결/분리에 대 한 셀 수 있도록 화학 물질의 ...

1961 년, Hayflick와 무어 헤드 문화에 기본 fibroblasts 연속 구절1후 그들의 증식 잠재력을 잃고 보고. 이 프로세스는 각 세포 분열 후 telomeres의 순차 단축에 의해 발생 합니다. Telomeres 비판적으로 짧은 길이 도달, 그들은 활성화 확산의 돌이킬 수 없는 체포 하는 DNA 손상 응답 (DDR)에 의해 인식 됩니다-또한 일차 노 쇠로 정의. 일차 노화 현재 구분 mitogens와 apoptotic 신호2,3셀을 렌더링 하는 영구 세포 주기 검거의 상태를 유도 하는 것으로 알려져 있습니다 많은 자극 중 하나입니다. 노화 프로그램은 일반적으로 높은 lysosomal 활동, 미토 콘 드 리아 기능 장애, 핵 변화, chromatin 재배열, 바인딩과 그물 스트레스, DNA 손상 및 노화 관련을 포함 하 여 추가 기능 특징 분 비 형 (SASP)3,4. 노화 세포는 신체에 여러 기능을가지고: 개발, 상처 치유 및 종양 억제2. 똑같이, 그들은 노화와, 역설적으로, 종양 진행5에서 중요 한 역할을 알려져 있습니다. 노화의 부정, 그리고 부분적으로 모순 된 효과 종종 SASP6에 기인 된다.
최근에, 그것은 생쥐에서 노화 세포의 수명 연장 하 고 노화 기능7,,89,10,11, 의 많은 것의 제거에 지도 표시 했다 12. 같은 방식으로, 여러 약물 개발 되었습니다 하거나 노화 세포 (senolytics)를 제거 하거나 대상 SASP13,14. 안티 에이징 치료 잠재력 최근 분야에 더 많은 관심을 모으고 있다.
세포 노화에 관련 된 메커니즘의 연구 그리고 약리학 내정간섭에 대 한 검 진 무 겁 게 비보 전 모델, 특히 인간의 기본 fibroblasts에 의존. 노화 표현 형에 있는 큰 가변성 관찰 이며 셀 유형, 자극 및 시간 포인트3,15, 등 다양 한 요인에 따라 다양 한 노화 inducers에 의해 활성화 하는 몇 가지 일반적인 기능을 확인 하 고는, 16,17. 공부 및 노화 세포를 대상으로 고려 하는 것이 필수적입니다. 따라서,이 프로토콜 일련의 다른 치료를 사용 하 여 기본 섬유 아 세포에서 노화를 유도 하는 데 사용 하는 메서드를 제공 하는 것을 목표로. 로 설명 될 것입니다 방법을 쉽게 다른 셀 형식에 적용할 수 있습니다.
일차 노화 외에도 다른 5 노화 유도 치료 설명: 이온화 방사선, 자외선 (UV) 방사선, 독 소 루비, 산화 긴장 및 epigenetic 변화 (즉 histone acetylation 또는 DNA demethylation의 진흥) . 둘 다, 이온화 방사선 및 자외선 직접적인 DNA 손상 그리고, 적절 한 복용량에 트리거 노화18,19. 독 소 루비 또한 DNA 손상을 통해 주로 노화 DNA로 intercalating 하 여 원인과 topoisomerase II 기능을 방해 하 고 따라서 중단 DNA 수리 메커니즘20. 노화에 대 한 필수적인 유전자의 표현은 일반적으로 histone acetylation와 DNA 메 틸 화에 의해 제어 됩니다. 결과적으로, DNA demethylating (예를 들어, 5-아 자) 에이전트 및 히스톤 deacetylase 억제제 (예를 들면, 나트륨 낙 산 염 및 사 하) 그렇지 않으면 정상 세포21,22노화를 트리거합니다.
마지막으로, 노화 세포에 관련 된 가장 일반적인 마커의 4 설명 될 것 이다:는 노화 관련-β-galactosidase (SA-β-gal) 듀 합동 분석 결과, 세포 주기 규칙의 overexpression에 의해 DNA 종합의 비율의 활동 및  종속 kinase 억제제 p16, p21, 및 overexpression 멤버는 SASP의 분 비

<table><tbody><tr><td>DMEM Media - GlutaMAX</td><td>Gibco</td><td>31966-047</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SV30160.03</td><td></td></tr><tr><td>Penicillin-Streptomycin (P/S; 10,000 U/ml)</td><td>Lonza</td><td>DE17-602E</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>SC-202581</td><td></td></tr><tr><td>Nuclease-Free Water (not DEPC-Treated)</td><td>Ambion</td><td>AM9937</td><td></td></tr><tr><td>T75 flask</td><td>Sarstedt</td><td>833911002</td><td></td></tr><tr><td>Trypsin/EDTA Solution</td><td>Lonza</td><td>CC-5012</td><td></td></tr><tr><td>PBS tablets</td><td>Gibco</td><td>18912-014</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Sigma-Aldrich</td><td>T9661-1000EA</td><td></td></tr><tr><td>Corning&amp;nbsp;15 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430791</td><td></td></tr><tr><td>6-well plate</td><td>Sarstedt</td><td>83.3920</td><td></td></tr><tr><td>24-well plate</td><td>Sarstedt</td><td>83.3922</td><td></td></tr><tr><td>13mm round coverslips</td><td>Sarstedt</td><td>83.1840.002</td><td></td></tr><tr><td>Steriflip</td><td>Merck Chemicals</td><td>SCGP00525</td><td></td></tr><tr><td>Cesium137-source</td><td></td><td></td><td>IBL 637 Cesium-137&amp;gamma;-ray machine</td></tr><tr><td>UV radiation chamber</td><td></td><td></td><td>Opsytec, Dr. G&amp;ouml;bel BS-02</td></tr><tr><td>Doxorubicin dihydrochloride&amp;nbsp;</td><td>BioAustralis Fine Chemicals</td><td>BIA-D1202-1</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>7722-84-1</td><td></td></tr><tr><td>5-aza-2&amp;rsquo;-deoxycytidine</td><td>Sigma-Aldrich</td><td>A3656</td><td></td></tr><tr><td>SAHA</td><td>Sigma-Aldrich</td><td>SML0061</td><td></td></tr><tr><td>Sodium Butyrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B5887</td><td></td></tr><tr><td>X-gal (5-Bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside)</td><td>Fisher Scientific</td><td>7240-90-6</td><td></td></tr><tr><td>Citric acid monohydrate</td><td>Sigma-Aldrich</td><td>5949-29-1</td><td></td></tr><tr><td>Sodium dibasic phosphate</td><td>Acros organics</td><td>7782-85-6</td><td></td></tr><tr><td>Potassium ferrocyanide&amp;nbsp;</td><td>Fisher Scientific</td><td>14459-95-1</td><td></td></tr><tr><td>Potassium ferricyanide</td><td>Fisher Scientific</td><td>13746-66-2</td><td></td></tr><tr><td>Sodium Chloride</td><td>Merck Millipore</td><td>7647-14-5</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Fisher Chemicals</td><td>7791-18-6</td><td></td></tr><tr><td>25% glutaraldehyde</td><td>Fisher Scientific</td><td>111-30-8,7732-18-5</td><td></td></tr><tr><td>16% formaldehyde (w/v)</td><td>Thermo-Fisher Scientific</td><td>28908</td><td></td></tr><tr><td>EdU (5-ethynyl-2&amp;rsquo;-deoxyuridine)</td><td>Lumiprobe</td><td>10540</td><td></td></tr><tr><td>Sulfo-Cyanine3 azide (Sulfo-Cy3-Azide)</td><td>Lumiprobe</td><td>D1330</td><td></td></tr><tr><td>Sodium ascorbate</td><td>Sigma-Aldrich</td><td>A4034</td><td></td></tr><tr><td>Copper(II) sulfate pentahydrate (Cu(II)SO&lt;sub&gt;4&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>209198</td><td></td></tr><tr><td>Triton X-100</td><td>Acros organics</td><td>215682500</td><td></td></tr><tr><td>TRIS base</td><td>Roche</td><td>11814273001</td><td></td></tr><tr><td>LightCycler 480 Multiwell Plate 384, white&amp;nbsp;</td><td>Roche</td><td>4729749001</td><td></td></tr><tr><td>Lightcycler 480 sealing foil&amp;nbsp;</td><td>Roche</td><td>4729757001</td><td></td></tr><tr><td>Sensifast Probe Lo-ROX kit&amp;nbsp;</td><td>Bioline</td><td>BIO-84020</td><td></td></tr><tr><td>UPL Probe Library</td><td>Sigma-Aldrich</td><td>Various</td><td></td></tr><tr><td>Human IL-6 DuoSet ELISA</td><td>R&amp;amp;D</td><td>D6050</td><td></td></tr><tr><td>Bio-Rad TC20</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Counting slides</td><td>Bio-Rad</td><td>145-0017</td><td></td></tr><tr><td>Dry incubator</td><td>Thermo-Fisher Scientific</td><td>Heratherm</td><td></td></tr><tr><td>Dimethylformamide</td><td>Merck Millipore</td><td>1.10983</td><td></td></tr><tr><td>Parafilm &#039;M&#039; laboratory film</td><td>Bemis&amp;nbsp;</td><td>#PM992</td><td></td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>Needles</td><td></td><td></td><td></td></tr></tbody></table>

induction and validation of cellular senescence in primary human cells

세포 노화의 영구 세포 주기 검거 다른 손상 자극에 반응에서 활성화 상태입니다. 세포 노화의 활성화는 다양 한 병 태 생리 조건 종양 억제, 조직 리 모델링 및 노화 등의 특징입니다. Vivo에서 세포 노화의 inducers는 아직도 가난 하 게 특징 이다. 그러나, 자극의 수 ex vivo세포 노화 촉진 사용할 수 있습니다. 그 중 가장 일반적인 노화-inducers 일차 피로, 이온화 및 비 이온화 방사선, 차적인 약물, 산화 스트레스, 그리고 demethylating와 acetylating 대리인 있습니다. 여기, 우리가 노화에 섬유 아 세포를 유도 하는 것이 자극을 사용 하는 방법에 자세한 지침을 제공 합니다. 이 프로토콜은 쉽게 1 차 셀 및 셀 라인, 암 세포를 포함 하 여 다른 유형의 적용할 수 있습니다. 우리는 또한 노화 유도의 유효성 검사를 위해 다른 방법을 설명합니다. 특히, 우리는 lysosomal 효소 노화 관련 된 β-galactosidase (SA-β-gal), 5-ethynyl-2'-deoxyuridine (듀) 합동 분석 결과, 세포 주기 식의 레벨을 사용 하 여 DNA 합성의 속도의 활동을 측정에 초점 억제제 p16, p21, 식 그리고의 Senescence-Associated 분 비 형 (SASP)의 분 비. 마지막으로, 우리 예제에서는 결과 제공 하 고 이러한 프로토콜의 응용 프로그램을 추가 논의.

SA-β-gal 노화 섬유 아 세포에서 얼룩의 농축
Β-galactosidase (β-gal)는 모든 세포에 표현 되 고 4.025,26의 최적 pH는 lysosomal 효소 이다. 그러나, 노화, 동안 리소좀 크기가 증가 하 고, 따라서, 노화 세포 축적 β-gal. 이 효소의 증가 금액 차선 pH 6.025,27...

Watch this Scientific Journal Video about Induction and Validation of Cellular Senescence in Primary Human Cells at JoVE.com

Induction and Validation of Cellular Senescence in Primary Human Cells

유도 및 기본 인간 세포에서 세포 노화의 유효성 검사

여기, 우리 유도 대 한 프로토콜의 시리즈 및 경작된 한 세포에 세포 노화의 유효성 검사를 설명합니다. 우리는 다른 노화 유도 자극에 집중 하 고 일반적인 노화 관련 마커의 정량화를 설명. 우리 모델, 섬유 아 세포를 사용 하 여 기술 정보를 제공 하지만 프로토콜은 다양 한 휴대 전화 모델에 적용할 수 있습니다.

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

induction-and-validation-of-cellular-senescence-in-primary-human-cells

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16.6K 조회수.  University of Groningen, University Medical Center Groningen. 여기, 우리 유도 대 한 프로토콜의 시리즈 및 경작된 한 세포에 세포 노화의 유효성 검사를 설명합니다. 우리는 다른 노화 유도 자극에 집중 하 고 일반적인 노화 관련 마커의 정량화를 설명. 우리 모델, 섬유 아 세포를 사용 하 여 기술 정보를 제공 하지만 프로토콜은 다양 한 휴대 전화 모델에 적용할 수 있습니다.

비디오: Induction and Validation of Cellular Senescence in Primary Human Cells

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

무 혈청의 수집 및 공급 장치없이 마우스 배아는 세포 자유로운 접근을위한 세포 조절 중간 줄기

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

연구

발생학

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.

부상-유도 노화 및 골격 근육에서 프로그래밍을 Vivo에서 의 평가

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

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.

노화 암 세포를 정량화하는 민감한 방법

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

의학

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.

Oncogene 유도 노화 중 반응성 산소 종 및 정상적인 인간 섬유 아 세포에서 분 비 형 노화 관련의 정량적 측정

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.

치료 유도 노인성 암 세포에서 다중 형광 노화 마커의 동시 이미징 및 유동 세포 측정 기반 검출

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

암 연구학

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.

유세포분석을 통한 노화 종양 세포의 식별 및 농축을 위한 원적색 형광 노화 관련 β-갈락토시다아제 프로브

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.

대사 표지 및 질량 분광법으로 노화 및 비분할 배양된 세포에서의 단백질 회전율 측정

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

생화학

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.

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.

기술은 유도 및 정량화 세포 노화하기

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 ...

생물학

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-갈락토시다아제 계 선별 분석법

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 ...