Name: strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis
Uploaded: 2015-02-16T13:00:00.000+00:00
Duration: 12 min 55 s
Description: Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

우리는 단어 &quot;아나스타시스&quot;세포 사멸의 반전을 설명하는 제안에 대해 목사 랄프 Bohlmann와 목사 제임스 Voelz 감사합니다; 안정적으로 시토크롬 C -GFP을 표현 헬라 세포에 대한 더글라스 R. 녹색; H446 세포 찰스 M. 루딘 에릭 E. 가드너; 비디오에서 만화 그리기에 도움을 헤더 어린 양; 이 원고의 가치있는 토론을위한 유 후이 여진. 이 작품은 선생님 에드워드 Youde 기념 원정대 (HLT), 박사 월터 제토 기념 장학금 (HLT), 풀 브라이트 부여 007-2009 (HLT), 생명 과학 연구 재단의 교제 (HLT)에 의해 지원되었다, NIH는 NS037402 (JMH) 부여 및 NS083373 (JMH), 그리고 홍콩의 대학 보조금위원회 광역 / B-07 / 99 (MCF). 호 램 당나라는 생명 과학 연구 재단의 Shurl와 케이 쿠 르치 재단 연구원이다.

The authors have nothing to disclose.

아나스타시스는 세포 사멸 경로를 활성화 한 세포가 계속해서 죽어가는 과정을 역 생존 현상을 의미한다. 여기서는 생균 촬상 사실 동일인 셀 늦은 단계에서 아폽토시스 세포 사멸의 과정을 역으로 한 후 생존 및 재생을 계속할 수 있음을 확인하기 위해 사용될 수 있다는 것을 증명하고있다. 우리 프로토콜은 아폽토시스를 유도 및 확인을위한 여러 가지 바이오 마커와 세포 사멸의 모니터링 반전...

세포 사멸 ( &quot;죽음에 떨어지는&quot;에 대한 그리스어)는 일반적으로 세포 자살 1-7로 끝나는 단방향 프로세스로 간주됩니다. 이미 세포 사멸 경로의 8,9을 시작했다 세포를 포함, 그렇지 않으면 전체 동물에 죽을 것 여분의 세포의 생존에 프로 죽음 유전자 결과의 유전 중단. 마찬가지로, 유전자 조작 인위적으로 표시 신호를 &quot;날 먹고&quot;또는 자신의 세포 외 기질 접착 성을 잃게 건강한 포유 동물 세포는 모든 세포의 식균 작용 또는 entosis 각각 10, 11에 의해 죽음을 탈출 할 수 있습니다. 그러나, 우리와 다른 사람들은 또한 세포 사멸 12-15의 초기 단계에서 복구 할 수 있습니다 유전자 조작 정상적인 건강한 포유 동물 세포 및 세포 선없이 것을 보여 주었다. 세포는 전형적인 중요 체크 포인트를 통과 한 후 각각의 세포를 추적 할 수있는 도구를 사용하여, 우리는 또한, 세포 사멸 (12, 13)의 후반 단계에서 복구를 증명하고있다해동 2-6 &quot;니나&quot;를 표시합니다. 최종 단계의 세포 사멸이 체크 포인트는 미토콘드리아 시토크롬 C의 릴리스, 카스파의 활성화, 핵 분열 및 세포 사멸 기관의 형성을 포함한다. 우리는 세포 사멸 2-6의 위기에서 세포 사멸이 반전을 설명하기 위해, &quot;생활에 상승&quot;을 의미 그리스어 합성어 &quot;아나스타시스&quot;을 채택했다. 전체 죽어 복구 프로세스가 라이브 세포 이미징에 의해 관찰되지 않는 한,이 세포 사멸 이벤트를 경험하지 못한 세포에서 아나스타시스를받은 세포를 구별하기 위해 도전하고있다. 작품의 수십 년 세포 사멸에 의한 세포 자살의 형태 적 특징은 진화 적으로 보존 된 생화학 및 분자 이벤트 16-19에 의해 구동되는 것을 밝혀졌다. 이러한 이벤트는 손상 또는 d 제거하여 단세포와 다세포 생물의 발달과 homoeostatic 과정을 조절하는 세포의 자기 파괴를 촉진angerous 세포 16-19. 사멸 세포에 쉽게 사멸 1,5,6,16,20의 표준화, 형태 학적, 생화학 및 분자 발현에 의해 구별 될 수 있지만, 현재 아나스타시스 12, 13에 해당 알려진 마커가 없습니다. 중요한 것은, 아나스타시스를받은 세포는 정상적인 건강한 세포, 단지 세포 사멸을 반대로 시작 세포 표시와 같은 세포 사멸 죽어가는 세포 (12, 13)로 나타납니다. 따라서, 새로운 도구는 주어진 생존 세포가 이전의 활성 세포 사멸 과정을 경험했다 확실하게 결론을 내릴 필요가있다. 이 신속하고 대규모 파괴 과정이기 때문에 세포 사멸은 일반적으로 돌이킬 수없는 계단식으로 가정한다. 미토콘드리아는 세포질 (21, 22)에 시토크롬 C와 같은 apoptogenic 요인을 발표 한 후에는 일부 세포가 세포 사멸을 시작하는 것이 일 분 정도 걸릴 수 있지만, 카스파는 5 분 23, 24 내에서 활성화 세포질 다음에 할 수 있고,핵 10 분 25 ~ 27 내에서 응결 및 세포 사멸 잠시 후 25-27. 활성화 된 카스파은 절단과 같은 엔도 뉴 클레아 제 억제제 DFF45 / ICAD 29,30로서 셀룰러 철거 2,28의 주요 목적 구조적 및 기능적 구성 요소를 비활성화하여 아폽토시스를 조율. 카스파는 또한 31, 32 크롬 c의 미토콘드리아 방출을 촉진하는 미토콘드리아에 옭 BCL-2 가족의 BID, 같은 프로 세포 사멸 인자를 활성화합니다. 카스파 활동은 또한 탐식 33 대 식세포 또는 인접 세포에 의한 세포 죽음의 말림을 촉진하기위한 &quot;날 먹고&quot;신호로 포스파티딜 세린의 세포 표면에 노출되는 결과를 초래한다. 또한, 세포 사멸 이벤트는 세포 생체 에너지 대사의 34,35,36를 방해하는 역기능 미토콘드리아는, 렌더링합니다. 따라서, 이러한 파괴를 복구 직관적 아닌 것 같습니다. 원래는 달리 기대관리 포인트, 세포도 늦은 단계에서 사멸 세포 사멸 과정을 취소 할 수 있습니다. 지속적으로 배양 세포를 죽음의 운명을 모니터링함으로써, 우리는 일차 전지 및 세포주 12, 13의 범위에서 후기의 세포 사멸의 가역성을 관찰했다. 죽음의 자극의 제거는, 이러한 미토콘드리아의 분열, 염색질 응축, DNA 손상, 세포막에 blebbing, 포스파티딜 세린의 세포 표면에 노출, 미토콘드리아 시토크롬 C의 릴리스, 카스파 제 활성화, 핵 분열, 세포의 수축으로 세포 사멸의 명백한 기능에서 복구를 허용 세포 사멸 몸과 형성. 이러한 관찰은 아나스타시스의 기능, 결과, 및 메커니즘에 관한 대답없는 질문을 올립니다. 이러한 질문을 해결하기 위해, 전제 조건은 안정적 아나스타시스를받은 세포를 식별하는 것입니다. 여기서는 라이브 현미경 방법 및 이전 말기 아폽토시스 번째 반전 한 세포를 검출하기위한 바이오 센서를 카스파 서술욕실은 살아 남았다.

<table><tbody><tr><td>LSM780 confocal microscopy</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Glass bottom culture dish</td><td>MatTek Corporation</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Transparent CultFoi</td><td>Carl Zeiss</td><td>000000-1116-084</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; independent medium</td><td>Life Technologies</td><td>18045-088</td><td></td></tr><tr><td>CellTracker</td><td>Life Technologies</td><td>C34552</td><td></td></tr><tr><td>Mitotracker Red CMXRos</td><td>Life Technologies</td><td>M-7512</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H1399</td><td></td></tr><tr><td>Fluorescently labeled annexin V</td><td>Biovision</td><td>K201</td><td></td></tr></tbody></table>

strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis

아나스타시스 ( &quot;생활에 상승&quot;에 대한 그리스어) 죽어가는 세포의 회복을 의미한다. 이들 세포의 회복 전에, 미토콘드리아 단편화, 세포질 내로 미토콘드리아 사이토 크롬 C의 방출, 카스파 제 활성화, 크로 마틴 응축, DNA 손상, 핵 분열, 세포막에 blebbing, 세포 수축, 세포 표면 노출을 포함한 세포 사멸 중요한 체크 포인트를 통과 한 포스파티딜 세린, 및 세포 사멸 기관의 형성. 세포 사멸 자극함으로써 죽어가는 세포가 사멸하고 잠재적으로 다른 죽음의 메커니즘을 취소 할 수 있도록, 죽음 이전에 제거 될 때 아나스타시스가 발생할 수 있습니다. 따라서, 아나스타시스는 부적절하게 손상된 세포를 유지할 수있는 생리적 치유 과정을 포함 나타납니다. 기능 및 메커니즘 아나스타시스 명백하게 건강한 세포의 복구 후에 과거 사건을 검출하기위한 제한된 도구에 의해 부분적으로 방해, 여전히 불분명하다. 전략 anasta를 검출SIS는 아나스타시스를 변조 생리 메커니즘 연구, 질병 병리 언데드 세포의 유해성과 잠재적 인 치료제를 가능하게 할 것이다. 여기서 우리는 살아있는 세포 현미경 및 포유 동물 세포에 아나스타시스를 식별하고 추적하기위한 포유 동물 카스파 바이오 센서를 사용하여 효과적인 전략을 설명합니다.

세포 사멸의 반전을 연구하기 위해, 조직 배양 세포를 처음으로 세포 사멸을 유발하는 죽음의 자극에 노출되어있다. 세포가 세포 사멸의 특징을 표시 할 때, 신선한 배양액 후 자극을 씻어 후 복구 (그림 1A)를 허용하도록 죽어가는 세포를 배양 적용됩니다. 여기서 다루고있는 핵심 문제는 개별 죽어 배양 된 세포가 세포 사멸을 향해 진행 여전히 아나스타시스을받을 수있는 방법까지?...

Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

추적 아나스타시스, 세포 사멸을 취소 세포 생존 현상에 대한 전략

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

strategies-for-tracking-anastasis-cell-survival-phenomenon-that

Research

JoVE Journal

Biology

18.0K 조회수.  Johns Hopkins University Bloomberg School of Public Health. The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis. 

비디오: Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

장기 라이브 셀 이미징 셀 운명 Paclitaxel에 대 한 응답에서을 평가 하

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

연구

암 연구학

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

새로 형성 된 유방암 줄기 세포와 같은 세포 Apoptosis 반전 후 흐름 Cytometric 감지

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

유세포측정에 의한 유독성 응력 후 γH2AX 및 세포자멸의 세포 주기별 측정

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

유리를 통해 보면 : 시간 ​​경과 현미경 및 단일 세포의 종 추적 항암 치료를 공부하기

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

의학

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Anastasis에서 Vivo에서 CaspaseTracker 바이오 센서에 의해 감지

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

태아의 프로그래밍 셀 죽음을 감지 할 LysoTracker의 이용 및 배아 줄기 세포를 차별화

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

생물학

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

E4orf4 - 유도 세포 사망에 최저 효과의 유전자 발현 및 측정의 조건부 최저위한 셀 라인의 작성

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

동기화를 필요로하지 않고 유동 세포 계측법을 사용하여 세포주기 진행의 시간적 추적

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

세포 내 이웃 셀을받은 사멸 세포 죽음과의 상호 작용에 의해 실행 가능한 세포에서 유도 된 이벤트를 시그널링의 식별

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

세포사에 대한 소개

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

추적 아나스타시스, 세포 사멸을 취소 세포 생존 현상에 대한 전략

요약

더 많은 비디오 탐색

이 비디오의 챕터

장기 라이브 셀 이미징 셀 운명 Paclitaxel에 대 한 응답에서을 평가 하

새로 형성 된 유방암 줄기 세포와 같은 세포 Apoptosis 반전 후 흐름 Cytometric 감지

유세포측정에 의한 유독성 응력 후 γH2AX 및 세포자멸의 세포 주기별 측정

유리를 통해 보면 : 시간 경과 현미경 및 단일 세포의 종 추적 항암 치료를 공부하기

Anastasis에서 Vivo에서 CaspaseTracker 바이오 센서에 의해 감지

태아의 프로그래밍 셀 죽음을 감지 할 LysoTracker의 이용 및 배아 줄기 세포를 차별화

E4orf4 - 유도 세포 사망에 최저 효과의 유전자 발현 및 측정의 조건부 최저위한 셀 라인의 작성

동기화를 필요로하지 않고 유동 세포 계측법을 사용하여 세포주기 진행의 시간적 추적

세포 내 이웃 셀을받은 사멸 세포 죽음과의 상호 작용에 의해 실행 가능한 세포에서 유도 된 이벤트를 시그널링의 식별

세포사에 대한 소개

추적 아나스타시스, 세포 사멸을 취소 세포 생존 현상에 대한 전략

요약

더 많은 비디오 탐색

이 비디오의 챕터

장기 라이브 셀 이미징 셀 운명 Paclitaxel에 대 한 응답에서을 평가 하

새로 형성 된 유방암 줄기 세포와 같은 세포 Apoptosis 반전 후 흐름 Cytometric 감지

유세포측정에 의한 유독성 응력 후 γH2AX 및 세포자멸의 세포 주기별 측정

유리를 통해 보면 : 시간 ​​경과 현미경 및 단일 세포의 종 추적 항암 치료를 공부하기

Anastasis에서 Vivo에서 CaspaseTracker 바이오 센서에 의해 감지

태아의 프로그래밍 셀 죽음을 감지 할 LysoTracker의 이용 및 배아 줄기 세포를 차별화

E4orf4 - 유도 세포 사망에 최저 효과의 유전자 발현 및 측정의 조건부 최저위한 셀 라인의 작성

동기화를 필요로하지 않고 유동 세포 계측법을 사용하여 세포주기 진행의 시간적 추적

세포 내 이웃 셀을받은 사멸 세포 죽음과의 상호 작용에 의해 실행 가능한 세포에서 유도 된 이벤트를 시그널링의 식별

세포사에 대한 소개

유리를 통해 보면 : 시간 경과 현미경 및 단일 세포의 종 추적 항암 치료를 공부하기