eoe sub articles

EoE Sub Articles

1. Maintenance of NPCs
<ol>
	<li>Plate NPCs at a density of 1.5 million cells into an ECM-mimic gel-coated well containing 2 mL of 100% Expansion Media.</li>
	<li>Incubate NPCs at 37 &#176;C in a moist environment with 5% CO2.</li>
	<li>Add 5 &#956;M of ROCK inhibitor to the media for NPCs at P3 or lower, or for thawed NPCs, to prevent excessive cell death. Change media after 24 h to remove ROCK inhibitor.</li>
	<li>Passage cells every 4&#8211;9 days, depending upon when they become confluent. Cells are considered confluent when they become a densely packed monolayer covering the entire bottom of the dish surface.</li>
	<li>Plate NPCs at a density of 1 to 1.5 million cells per one well of a 6-well plate. Remove spent media every 48 h and replace with 2 mL of 100% Expansion Media.</li>
</ol>
3. Neural Precursor Cell DNA Synthesis Assay
<ol>
	<li>Plate 100,000 cells/well in a 24-well plate and assess in triplicate/condition.</li>
	<li>Add radioactive, tritiated [3H]-thymidine to the culture medium (1.5 &#956;Ci/mL) in each well after 46 h in culture. Incubate cells at 37 &#176;C for 2 h. 
	NOTE: When using radioactive materials, receive training from your institution, follow radioactivity safety protocols, and dispose of radioactive materials in the appropriately designated waste receptacles.</li>
	<li>Remove and properly dispose of radioactive media after 2 h. Add 300 &#956;L of pre-warmed 0.25% trypsin-EDTA (0.5 mM) to each well and incubate for 20 min at 37 &#176;C.</li>
	<li>Turn on the cell harvester (see Materials section) and the pump and ensure that the pump pressure is below 200 PSI. Place filter paper through the space in the cell harvester and tear off the right corner to mark paper orientation.</li>
	<li>Place collecting tubes of cell harvester into an empty &#34;blank&#34;&#160;tray and press prewash to moisten the filter paper. Place collecting tubes into sample wells and run (start).</li>
	<li>When the cell harvester has finished collecting samples, lift clamp and advance filter paper to repeat for all sample sets. Always prewash in an empty, &#34;blank&#34;&#160;plate.</li>
	<li>Dry filter paper under a light source and set up corresponding vials in a tray. Punch out paper chads into vials and add 2 mL of liquid scintillation cocktail to each vial. Cap and label vials.</li>
	<li>Incubate vials in a liquid scintillation cocktail for at least 1 h before reading the counts per minute (CPMs) on a scintillation machine.</li>
</ol>

dna synthesis assay: a technique to assess dna synthesis in proliferating cells using radioactive tritiated thymidine incorporation

Source: Williams, M. et al. <a href="https://www.jove.com/v/56628/rapid-detection-neurodevelopmental-phenotypes-human-neural-precursor">Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs).</a> J. Vis. Exp. (2018)
In this video, we determine the proliferative capacity of neuronal progenitor cells by measuring the incorporation of radioactive tracer tritiated thymidine into the DNA of cells. This technique can be used to study cell proliferation after treatment with certain drugs and growth factors.

DNA Synthesis Assay: A Technique to Assess DNA Synthesis in Proliferating Cells Using Radioactive Tritiated Thymidine Incorporation

Source: Williams, M. et al. Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs). J. Vis. Exp. (2018)
In this video, we ...

Begin by taking a multi-well plate containing adherent cells.
Treat these cells with a media containing tritiated thymidine - a radioactive version of thymidine -&#160; and incubate.
During incubation, actively replicating cells incorporate radioactive thymidine. This thymidine pairs opposite adenosine, generating radiolabeled DNA in proliferating cells.
Remove the spent media from wells. Add a lysis buffer to lyse the cells and release DNA into the suspension.
Prepare the cell harvester assembly by placing the collecting tubes of the harvester over the wells of the microplate.
Apply suction pressure to enable the aspiration of suspended DNA from the wells. This DNA deposits on the filter paper, forming specific spots.
Cut out the filter paper chads with DNA spots. Transfer these chads individually into scintillation vials.
Overlay the filter paper with a scintillation cocktail. Place the vial in a liquid scintillation counter.
Inside the counter, the labeled DNA emits energy by radioactive decay. This energy is absorbed by the components of the scintillation cocktail. Eventually, the cocktail components begin to transfer the pulses, which in turn are absorbed by the sensor.
The amount of radioactivity detected determines the extent of cell division.

dna-synthesis-assay-technique-to-assess-dna-synthesis-proliferating

Research

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

In vitro studies

1.0K Views. Source: Williams, M. et al. Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs). J. Vis. Exp. (2018) In this video, we determine the proliferative capacity of neuronal progenitor cells by measuring the incorporation of radioactive tracer tritiated thymidine into the DNA of cells. This technique can be used to study cell proliferation after treatment with certain drugs and growth factors. 

Video: DNA Synthesis Assay: A Technique to Assess DNA Synthesis in Proliferating Cells Using Radioactive Tritiated Thymidine Incorporation - Concept

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

JoVE Journal

Biology

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

DNA Synthesis Assay: A Technique to Assess DNA Synthesis in Proliferating Cells Using Radioactive Tritiated Thymidine Incorporation

Transcript