We are grateful to the E. coli stock center for MG1693; Wormbase; the Caenorhabditis Genetics Center which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40OD010440) for strains; Zach Pincus for statistical advice; Aiping Feng for reagents; Luke Schneider, Andrea Scharf, Sandeep Kumar, and John Brenner for training, advice, support, and helpful discussion; and the Kornfeld and Schedl labs for feedback on this manuscript. This work was supported in part by National Institutes of Health [R01 AG02656106A1 to KK, R01 GM100756 to TS] and a National Science Foundation predoctoral fellowship [DGE-1143954 and DGE-1745038 to ZK]. Neither the National Institutes of Health nor the National Science Foundation had any role in the design of the study, collection, analysis, and interpretation of data, nor in writing the manuscript.

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The authors have nothing to disclose.

Preparation of EdU-labeled bacteria (step 1) is critical for this protocol, and the first point for troubleshooting. Wild-type young adult hermaphrodites label very reliably in a 4 h EdU-pulse, making this a useful control for every new batch of EdU-labeled bacteria. Additionally, intact EdU-labeled bacteria that enter the intestine (in older animals or certain pharynx/grinder defective mutants) will label with click chemistry and appear as bright oblong puncta in the gut. An alternative technique for labeling hermaphrodites uses a &#8220;soak&#8221; in a high concentration (1 mM) of EdU3. This technique starves the animals for the duration of labeling, but provides a useful way to bypass making EdU-labeled bacteria when troubleshooting fixation and click chemistry. If an EdU &#8220;soak&#8221; experiment is successful while an EdU feed is not, then prepare fresh EdU-labeled bacteria. To reach a sufficient bacterial density while also achieving a high EdU content, one may need to adjust the concentrations of EdU and thymidine.
The main limitation of this technique for labeling of S-phase is in the need to feed EdU-labeled bacteria to animals. The animals that cannot feed (due to genotype or stage) may not be labeled with this technique. Nevertheless, nucleoside analogs are currently the only method to identify S-phase nuclei in the C. elegans germline, and their use does not require that any transgenes be present in the animals. Additionally, once incorporated, EdU remains in nuclei even as they exit S-phase, progress through the cell cycle, divide, or differentiate. The signal weakens by half with every cell division. This makes EdU perfect for tracking a cell&#8217;s history even through a few cell divisions.
The stability of EdU makes pulse-chase experiments straightforward; simply rinse excess EdU bacteria from the animals after the desired duration pulse is finished and transfer the animals to unlabeled bacteria. EdU remains in DNA and remains visible even after multiple cell divisions. However, the experiments are limited to a single type of S-phase label (a single pulse of EdU). Co-labeling with EdU and BrdU is possible in mammalian cells31 but has not been reported in C. elegans. Co-labeling of IdU and CldU is used in mammals32 but also has not been reported in C. elegans.
The main advantages of EdU labeling are that the method requires no transgenes, EdU can be fed to C. elegans during regular culture, the chemistry is compatible with immunofluorescent techniques, and EdU persists in DNA for a long time after feeding has stopped. These features make EdU a great tool to study many aspects of the cell cycle and germ cell dynamics.
Cell cycle and germ cell dynamics analysis with EdU can be applied to a variety of research questions. Just a few examples of further applications of this method: How do the dynamics of the cell cycle change in animals with cell cycle gene mutations? How do physiological conditions affect the cell cycle in stem cells, the rate of germ cell entry into meiotic prophase, and the rate of germ cell progression through meiotic prophase? How does the cell cycle change during larval development? How do major signaling pathway disruptions affect the cell cycle, in addition to changes in cell fate (such as ectopic proliferation)? This system can be modified to study what the cells are doing in many different conditions.

In animal development, hundreds, thousands, millions, billions, or even trillions of cell divisions are required to form the adult organism. The cell cycle, the set of cellular events composed of G1 (gap), S (synthesis), G2 (gap), and M (mitosis) define the series of events that are executed each cell division. The cell cycle is dynamic and best appreciated in real time, which can be technically difficult. The techniques presented in this protocol allow one to make the measurements of the phases and timing of the cell cycle from still images.
Labeling with nucleoside analogs such as 5-ethynyl-2&#39;-deoxyuridine (EdU) or 5-bromo-2&#39;-deoxyuridine (BrdU) is the gold standard to identify S-phase in the studies of cell cycle dynamics in the Caenorhabditis elegans (C. elegans) adult hermaphrodite germline1,2,3,4,5. Both EdU and BrdU can be used in nearly any genetic background, as they do not rely on any genetic construct. Visualizing BrdU requires harsh chemical treatment to expose the antigen for anti-BrdU antibody staining, which is often incompatible with the assessment of other cellular markers visualized by co-staining with additional antibodies. By contrast, visualizing EdU occurs by click chemistry under mild conditions and thus is compatible with antibody co-staining6,7.
The specificity of the label is clear, since nuclei only incorporate the thymidine (5-ethynyl-2&#39;- deoxyuridine) analogs into DNA during S-phase. Visualization takes place in fixed tissue. The EdU label is invisible by itself until an azide-containing dye or fluorophore reacts covalently with the alkyne in EdU by copper-catalyzed click chemistry8. EdU labeling can provide immediate information on which nuclei are in S-phase, using a short pulse of labeling. EdU can also provide dynamic information, using pulse-chase or continuous labeling; for example, in a pulse-chase experiment, the label is diluted at each cell division or propagated as nondividing cells progress through development.
The C. elegans hermaphrodite germline is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle. The adult germline is a polarized assembly-line with stem cells found at the distal end followed by entry and progression through meiotic prophase, coordinated with the stages of gametogenesis more proximally (Figure 1). At the proximal end, oocytes mature, are ovulated and fertilized and begin embryogenesis in the uterus9,10,11. The ~20 cell-diameter long region near the distal tip cell, which includes the mitotically cycling germline stem, progenitor cells and meiotic S-phase cells but not the cells in meiotic prophase, is called the progenitor zone2,4,9,12. The cell membranes provide incomplete separation between the nuclei in the distal germline, but the progenitor zone cells undergo mitotic cell cycling largely independently. The median mitotic cell cycle duration of germline progenitor zone cells in young adult hermaphrodites is ~6.5 h; G1 phase is brief or absent, and quiescence is not observed1,2,13. Germline stem cell differentiation occurs through essentially direct differentiation and thus lacks transit-amplifying divisions4. During differentiation in the pachytene stage, approximately 4 out of 5 nuclei will not form oocytes but instead undergo apoptosis, acting as nurse cells by donating their cytoplasmic contents to the developing oocyte12,14,15.
In addition to labeling cells in S-phase with nucleoside analogs, one can identify the cells in mitosis and meiosis using antibody staining. Nuclei in mitosis are immunoreactive to anti-phospho-histone H3 (Ser10) antibody (called pH3)7,16. Nuclei in meiosis are immunoreactive to anti-HIM-3 antibody (a meiotic chromosome axis protein)17. Nuclei in the progenitor zone can be identified by the absence of HIM-3, the presence of nucleoplasmic REC-818, or the presence of WAPL-119. WAPL-1 intensity is highest in the somatic gonad, high in the progenitor zone, and low during early meiotic prophases19. Several cell cycle measurements are possible with a few variations in the protocol: I) identify nuclei in S-phase and measure S-phase index; II) Identify nuclei in M-phase and measure the M-phase index; III) determine whether nuclei were in mitotic or meiotic S-phase; IV) measure the duration of G2; V) measure the duartion&#160;of G2+M+G1 phases; VI) measure the rate of meiotic entry; VII) estimate the rate of meiotic progression.
One can make multiple cell cycle measurements from only a few types of wet-lab experiments. The protocol below describes a 30 min pulse labeling by feeding C. elegans adult hermaphrodites with EdU labeled bacteria and co-labeling M-phase cells by staining with anti-pH3 antibody and progenitor zone cells by staining with anti-WAPL-1 antibody. Only changes in the duration&#160;of EdU feed (Step 2.5), type of antibodies employed (Step 5), and analyses (Step 8.3) are required for the additional measurements.

<table><tbody><tr><td>&lt;em&gt;E. coli MG1693&lt;/em&gt;</td><td>Coli Genetic Stock Center</td><td>6411</td><td>grows fine in standard unsupplemented LB</td></tr><tr><td>&lt;em&gt;E. coli OP50&lt;/em&gt;</td><td>Caenorhabditis Genetics Center</td><td>OP50</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Imaging Kit</td><td>Thermo Fisher Scientific</td><td>C10337</td><td></td></tr><tr><td>5-Ethynyl-2&amp;prime;-deoxyuridine</td><td>Sigma</td><td>900584-50MG</td><td>or use EdU provided in kit</td></tr><tr><td>Glucose</td><td>Sigma</td><td>D9434-500G</td><td>D-(+)-Dextrose</td></tr><tr><td>Thiamine (Vitamin B1)</td><td>Sigma</td><td>T4625-5G</td><td>Reagent Grade</td></tr><tr><td>Thymidine</td><td>Sigma</td><td>T1895-1G</td><td>BioReagent</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>M1880-1KG</td><td>MgSO4, Reagent Grade</td></tr><tr><td>Sodium Phosphate, dibasic, anhydrous</td><td>Fisher</td><td>BP332-500G</td><td>Na2HPO4</td></tr><tr><td>Potassium Phosphate, monobasic</td><td>Sigma</td><td>P5379-500G</td><td>KH2PO4</td></tr><tr><td>Ammonium Chloride</td><td>Sigma</td><td>A4514-500G</td><td>NH4Cl, Reagent Plus</td></tr><tr><td>Bacteriological Agar</td><td>US Biological</td><td>C13071058</td><td></td></tr><tr><td>Calcium Chloride dihydrate</td><td>Sigma</td><td>C3881-500G</td><td>CaCl</td></tr><tr><td>LB Broth (Miller)</td><td>Sigma</td><td>L3522-1KG</td><td>Used at 25g/L</td></tr><tr><td>Levamisole</td><td>Sigma</td><td>L9756-5G</td><td>0.241g/10ml</td></tr><tr><td>Phosphate buffered saline</td><td>Calbiochem Omnipur</td><td>6506</td><td>homemade PBS works just as well</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379-500ML</td><td></td></tr><tr><td>16% Paraformaldehyde, EM-grade ampules</td><td>Electron Microscopy Sciences</td><td>15710</td><td>10ml ampules</td></tr><tr><td>100% methanol</td><td>Thermo Fisher Scientific</td><td>A454-1L</td><td>Gold-label methanol is critical for proper morphology with certain antibodies</td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-072</td><td>Lot 1671330</td></tr><tr><td>rabbit-anti-WAPL-1</td><td>Novus biologicals</td><td>49300002</td><td>Lot G3048-179A02, used at 1:2000</td></tr><tr><td>mouse-anti-pH3 clone 3H10</td><td>Millipore</td><td>05-806</td><td>Lot#2680533, used at 1:500</td></tr><tr><td>goat-anti-rabbit IgG-conjugated Alexa Fluor 594</td><td>Invitrogen</td><td>A11012</td><td>Lot 1256147, used at 1:400</td></tr><tr><td>goat-anti-mouse IgG-conjugated Alexa Fluor 647</td><td>Invitrogen</td><td>A21236</td><td>Lot 1511347, used at 1:400</td></tr><tr><td>Vectashield antifade mounting medium containing 4&#039;,6-Diamidino-2-Phenylindole Dihydrochloride (DAPI)</td><td>Vector Laboratories</td><td>H-1200</td><td>mounting medium without DAPI can be used instead, following a separate DAPI incubation</td></tr><tr><td>nail polish</td><td>Wet n Wild</td><td>DTC450B</td><td>any clear nail polish should work</td></tr><tr><td>S-medium</td><td>various</td><td></td><td>see wormbook.org for protocol</td></tr><tr><td>M9 buffer</td><td>various</td><td></td><td>see wormbook.org for protocol</td></tr><tr><td>M9 agar</td><td>various</td><td></td><td>same recipe as M9 buffer, but add 1.7% agar</td></tr><tr><td>Nematode Growth Medium</td><td>various</td><td></td><td>see wormbook.org for protocol</td></tr><tr><td>dissecting watch glass</td><td>Carolina Biological</td><td>42300</td><td></td></tr><tr><td>Parafilm laboratory film</td><td>Pechiney Plastic Packaging</td><td>PM-996</td><td>4 inch wide laboratory film</td></tr><tr><td>petri dishes</td><td></td><td></td><td>60 mm diameter</td></tr><tr><td>Long glass Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>1ml centrifuge tubes</td><td>MidSci Avant</td><td>2926</td><td></td></tr><tr><td>Tips</td><td></td><td></td><td></td></tr><tr><td>Serological pipettes</td><td></td><td></td><td></td></tr><tr><td>500 mL Erlenmyer flask</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>25G 5/8&amp;rdquo; needles</td><td>BD PrecisionGlide&amp;nbsp;</td><td>305122</td><td></td></tr><tr><td>5ml glass centrifuge tube</td><td>Pyrex&amp;nbsp;</td><td></td><td></td></tr><tr><td>Borosilicate glass tubes 1ml</td><td></td><td></td><td></td></tr><tr><td>glass slides</td><td></td><td></td><td></td></tr><tr><td>no 1 coverslips 22 x 40 mm</td><td></td><td></td><td>no 1.5 may work, also</td></tr><tr><td>37 &amp;deg;C Shaker incubator</td><td></td><td></td><td></td></tr><tr><td>Tabletop Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Clinical Centrifuge</td><td>IEC</td><td>428</td><td>with 6 swinging bucket rotor</td></tr><tr><td>Mini Centrifuge</td><td></td><td></td><td></td></tr><tr><td>20 &amp;deg;C incubator</td><td></td><td></td><td></td></tr><tr><td>4 &amp;deg;C refrigerator</td><td></td><td></td><td></td></tr><tr><td>-20 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>Observer Z1 microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Plan Apo 63X 1.4 oil-immersion objective lens</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Ultraview Vox spinning disc confocal system</td><td>PerkinElmer&amp;nbsp;</td><td></td><td>Nikon spinning disc confocal system works very well, also, as described here: http://wucci.wustl.edu/Facilities/Light-Microscopy&amp;nbsp;</td></tr></tbody></table>

cell cycle analysis in the c. elegans germline with the thymidine analog edu

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Watch this Scientific Journal Video about Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU at JoVE.com

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

cell-cycle-analysis-c-elegans-germline-with-thymidine-analog

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.3K Views.  Washington University School of Medicine, St. Louis, Missouri. Deze methode kan helpen bij het beantwoorden van belangrijke vragen over de celcyclus en kiemlijn stamcel populatiedynamiek in C.elegans. De belangrijkste voordelen van deze techniek zijn dat het geen transgenes vereist, het is compatibel met immunofluorescente kleuring, en het kan worden gewijzigd om cellen te bestuderen onder veel verschillende omstandigheden. Ik was erg opgewonden toen ik voor het eerst zag deze methode wordt gebruikt in de Scheda lab.Hoewel deze methode inzicht kan...

Video: Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Genetics

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Neuroscience

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 ...

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 ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S&gt;G2/M (progression from S to G2/M), and M phases.

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Cell cycle analysis with 5-ethynyl-2'-deoxyuridine (EdU) and phospho-histone H3 (pH3) labeling is a multi-step procedure that may require extensive optimization. Here, we present a detailed protocol that describes all steps for this procedure including image analysis and quantification to distinguish cells in different cell cycle phases.

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) ...

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome segregation. As DNA synthesis defects underlie chromosome rearrangements, monitoring DNA replication has become essential to understand the basis of genome instability. Saccharomyces cerevisiae is a classical model to study cell cycle regulation, but key DNA replication parameters, such as the fraction of cells in the S phase or the S-phase duration, are still difficult to determine. This protocol uses short and non-toxic pulses of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, in engineered TK-hENT1 yeast cells, followed by its detection by Click reaction to allow the visualization and quantification of DNA replication with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry. This method may identify previously overlooked defects in the S phase and cell cycle progression of yeast mutants, thereby allowing the characterization of new players essential for ensuring genome stability.

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

We describe two complementary protocols to accurately determine S-phase duration in S. cerevisiae using EdU, a thymidine analog, which is incorporated in vivo and detected using Click chemistry by microscopy and flow cytometry. It allows for the easy characterization of the duration of DNA replication and overlooked replication defects in mutants.

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

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