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Specific Cell Cycle Synchronization with Butyrate and Cell Cycle Analysis

Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in Madin Darby Bovine Kidney (MDBK) cells. We explore the possibility of using butyrate-blocked cells to obtain synchronized cells and we c ...

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Chromosome Formation During Fertilization in Eggs of the Teleost Oryzias latipes

Upon fertilization, eggs shift their cell cycle from the meiotic to the mitotic pattern for embryogenesis. The information on chromosome formation has been accumulated by various experiments using inhibitors to affect formation and behavior of chromosomes in the cycle of cell prolif ...

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Cell Synchronization by Inhibitors of DNA Replication Induces Replication Stress and DNA Damage Response: Analysis by Flow Cytometry

Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G2, and M. We ...

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Synchronization of Mammalian Cell Cultures by Serum Deprivation

Mammalian cells are amenable to study the regulation of cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for ...

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Detecting Recruitment of DNA Damage Response Factors Through the eChIP Approach

DNA interstrand crosslinks (ICLs) are lesions that covalently link the two strands of DNA. This type of DNA damage represents one of the most complex DNA lesions whose repair mechanisms remain largely unclear. Uncovering proteins involved in the processing of ICLs and understand how they in ...

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Indirect Immunofluorescence for Monitoring Spindle Assembly and Disassembly in Yeast

In yeast like all eukaryotes, microtubules are a crucial element of the mitotic spindle that separates the genetic material during cell division. The assembly status and position of the mitotic spindle, as well as cytoplasmic microtubules, can be monitored easily using indirect immunof ...

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Assessing G1-to-S-Phase Progression After Genotoxic Stress

Maintenance of genomic integrity is critical for the survival of organisms. Thus, mammalian cells employ a complex DNA damage response that can sense and repair DNA damage. One important aspect of the cellular DNA damage response is the activation of checkpoints that result in cell cycle arre ...

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Analyzing p53 Regulated DNA Damage Checkpoints by Flow Cytometry

The most critical feature of the cellular response to DNA damage is the ability of the cell to pause and repair the damage so that detrimental mutations will not be passed along to future generations of cells. The cell cycle of mammalian cells is equipped with checkpoints that can prevent cell cycle pro ...

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Using Drosophila S2 Cells to Measure S phase-Coupled Protein Destruction via Flow Cytometry

Cell proliferation depends on the timely synthesis and destruction of proteins at specific phases of the cell cycle. Recently it was discovered that the destruction of several key cell cycle regulatory proteins during S phase is coupled directly to DNA replication. These proteins harbor a m ...

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A Human Cell Extract-Based Assay for the Activation of ATM and ATR Checkpoint Kinases

The Ataxia telangiectasia-mutated (ATM) and the ATM-Rad3-related (ATR) kinases are master regulators of the DNA damage-signaling pathways that respond to a wide variety of DNA damage. In this chapter, we describe an in vitro biochemical assay to study the activation of ATM and ATR by double-st ...

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Methods for Studying Checkpoint Kinases Chk1

Attempts to passage through mitosis with unrepaired DNA damage or incompletely replicated DNA leads to genome instability and/or cell death. To prevent this from occurring, an ancient checkpoint (known as the G2 DNA damage checkpoint) that inhibits the activation of the mitotic cyclin- ...

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Analyzing DNA Replication Dynamics of Genotoxin-Treated Cells Using Velocity Sedimentation

Following acquisition of DNA damage S-phase progression may potentially be affected via multiple mechanisms. For example DNA damage-activated signal transduction pathways negatively regulate the initiation of DNA synthesis at unfired origins of replication, a process term ...

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Study of Cell Cycle Checkpoints Using Xenopus Cell-Free Extracts

Cell cycle checkpoints are involved in the coordinated response to DNA damage and thus play a key role in maintaining genome integrity. Several model systems have been developed to study the mechanisms and complexity of checkpoint function. Here we describe the application of cell-free ex ...

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Methods to Study Cancer Therapeutic Drugs That Target Cell Cycle Checkpoints

Cell cycle checkpoints operating through a network of multiple signaling pathways provide a key mechanism for self-defense of cells against DNA damage caused by various endogenous or environmental stresses. In cancer treatment, checkpoints are activated in response to diverse DNA ...

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Studying S-Phase DNA Damage Checkpoints Using the Fission Yeast Schizosaccharomyces pombe

Slowing of replication in response to DNA damage is a universal response to DNA damage during S-phase. Originally discovered to be defective in checkpoint mutant cells in metazoans, this S-phase DNA damage checkpoint response has been extensively studied in yeast. Unlike other checkpoi ...

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Studying G2 DNA Damage Checkpoints Using the Fission Yeast Schizosaccharomyces pombe

Using synchronized cells, one can directly measure delay in mitosis brought about by the G2 DNA damage checkpoint in response to exposure to exogenous DNA damaging agents. Scoring mitosis in the fission yeast Schizosaccharomyces pombe is relatively simple. Many techniques exist for sy ...

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Evaluating Spindle Assembly Checkpoint Competence in Mouse Oocytes Using Immunoblotting

The spindle assembly checkpoint (SAC) is a quality control mechanism for overseeing the fidelity of chromosome segregation. By modulating the activity of the anaphase-promoting complex or cyclosome (APC/C), the SAC sets the timing of anaphase-onset by co-ordinating the timely destr ...

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Methods for Studying the G2 DNA Damage Checkpoint in Mammalian Cells

In response to post-replicative DNA damage, cells activate the G2 DNA damage checkpoint to ensure mitosis is not attempted until the damage has been repaired. This is a common response to a variety of DNA damaging agents, including ionizing radiation and many chemotherapeutic agents used in the ...

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Analysis of Changes in Protein Level and Subcellular Localization During Cell Cycle Progression Using the Budding Yeast Saccharomyces cerevisiae

Methods are described here to monitor changes in protein level and subcellular localization during the cell cycle progression in the budding yeast Saccharomyces cerevisiae. Cell synchronization is achieved by an α-factor-mediated block-and-release protocol. Cells are colle ...

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Studying Cell Cycle Checkpoints Using Drosophila Cultured Cells

Drosophila cell lines are valuable tools to study a number of cellular processes, including DNA damage responses and cell cycle checkpoint control. Using an in vitro system instead of a whole organism has two main advantages: it saves time and simple and effective molecular techniques are ava ...

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