We are grateful to George Jarvis,&#160;Martha Bock, and Bette Hecox-Lea, Marine Biological Laboratory, for their help in filming.

1. Preparation of Media
Note: Use half-diluted Brujewicz artificial seawater of salinity 16.5 ppt (PSU). Other artificial seawaters are also frequently used to culture Brachionus rotifers 25,26.
<ol>
	<li>Add 454 mM NaCl, 26 mM MgCl2, 27 mM MgSO4, 10 mM KCl, and 10 mM CaCl2 to 4.5 L of distilled water (final volume will be 5 L). Alternatively, use deionized dilution water instead of distilled water. Add CaCl2 after dissolving all other salts.</li>
	<li>Prepare the 0.48 M NaHCO3 stock solution (200x concentration). Add 25 ml of it to the above solution. The final concentration of NaHCO3 is 2.4 mM.</li>
	<li>Prepare the 0.4 M NaBr stock solution (500x concentration). Add 10 ml of it to the above solution. The final concentration of NaBr is 0.8 mM. Make up to 5 L with distilled water.</li>
	<li>Filter the solution with a 0.45 &#181;m membrane filter. Dilute it two times with sterile water before use (v/v). 
	Note: It is possible to make a 2x concentration of Brujewicz artificial seawater as a stock solution.</li>
</ol>
2. General Culture Conditions
<ol>
	<li>Culture laboratory-raised or wild-captured rotifers in a sterile 100 ml beaker between 20 and 30 &#176;C. Higher temperatures result in shorter lifespans and accelerated reproduction. Use 25 &#176;C for the convenience of experiments. The rotifer density or volume of artificial seawater is not a big issue here.</li>
	<li>Culture dietary microalgae species in artificial seawater 11. See Snell et al. (2014) for details 11. Typically, use Tetraselmis tetrathele (~2 x 105 cells/ml 27,28), T. suecica (~6 x 105 cells/ml 25,29), and Nannochloropsis oculata (~7 x 106 cells/ml 30,31). Since diet algae (species, culture conditions, biochemical compositions) significantly influences survival time of Brachionus rotifers, use the same lot of microalga in all experimental groups.</li>
	<li>Keep a stock population of rotifers in a batch culture by feeding and changing media occasionally (e.g., feed every 2 days and inoculate every week). Many neonates and adults bearing 2-3 eggs can be observed when rotifers are in optimal conditions. 
	Note: An alternative way to obtain experimental rotifers is to hatch resting eggs. Rotifers hatched from resting eggs are considered to be well synchronized, and their lifespan is not significantly different from that of rotifers hatched from amictic eggs 11. However, rotifers from resting eggs start reproduction earlier than those from amictic eggs. Thus, caution is needed for measurements of their reproductive traits.</li>
</ol>
3. Synchronization of Rotifers by Pre-culture
<ol>
	<li>Select a single rotifer from the stock population and culture as described in 2.1-2.3 to establish a sub-population that will be used for experiments. Typically culture for two weeks.</li>
	<li>Collect egg-bearing rotifers from the sub-population (collect a double number of individuals that will be used for experiment). Culture them as a single cohort (density: ~50 individuals/ml) in a 6-well culture plate as described in 2.1-2.3 in a fresh media under ad libitum feeding. Controlling population density is important because the conditioned medium affects reproductive physiology of Brachionus rotifers 32,33.</li>
	<li>Transfer neonates hatched from the adults&#39; first eggs to newly prepared culture media. Repeat this procedure over 2-3 generations.</li>
	<li>Use neonates hatched within a certain time period (e.g., &#60;3 hr) for measuring the survival time. To avoid a possible bias in selecting the single individual, although very improbable, check reproducibility using several independent sub-populations. 
	Note: As rotifers usually reproduce only asexually under this condition, ensure no males are present throughout the experiment. Males are smaller than neonates and typically move faster than females. Mictic females have different lifespans from amictic females under certain conditions 29.</li>
</ol>
4. Measurements of Survival Time
<ol>
	<li>Place neonates in plastic plates (typically 24- or 48-well plates, with each well containing 1 ml of artificial seawater).</li>
	<li>At 24 hr intervals, transfer the rotifers to newly prepared culture media or at 12 hr intervals if at higher temperature (30 &#176;C or above). Record the number of offspring and whether each individual is dead or alive. Record the rotifer as dead when cilia movement of the corona has completely stopped. 
	Note: Rotifers often attach to the side walls of the wells. Gentle pipetting of the water helps to find them. If rotifers are not found or are accidentally damaged by the pipetting, record them as &#34;censored&#34;, not as &#34;dead&#34;.</li>
	<li>Remove neonates when experimental rotifers are actively reproducing. Neonates grow rapidly and it is sometimes difficult to distinguish them from experimental rotifers.</li>
</ol>
5. Data Analysis
<ol>
	<li>Create the Kaplan-Meier survival curve (Figures 1 and 2) by plotting cumulative survival rate on the Y axis and time on the X axis. This is the most common representation of survival data. Use the non-parametric log-rank test (also called Mantel-Cox test) for statistical comparison of survival time 34. The log-rank test is also included in other standard statistical packages such as JMP and R. 
	Note: Do not use Student&#39;s t-test or analysis of variance (ANOVA) followed by parametric multiple comparison because normal distribution is usually not met by survival data 35. Also, these methods do not take censored individuals into account. Mann-Whitney U test can be used if there is no censored data.</li>
</ol>

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The authors have nothing to disclose.

The current protocol describes a method for measuring the survival time in Brachionus rotifers. The critical step is the synchronization of rotifer conditions over several generations. When experimental rotifers are well synchronized, a typical type I survival curve is observed with very little early mortality as reported in several previous studies 18,24,37,38. Standard deviations of their survival time therefore become smaller compared to poorly synchronized rotifers, resulting in high statistical power. Synchronization is also expected to increase reproducibility of survival time measurements&#160;&#8211; because mothers are cultured under optimal conditions, the current protocol offsets possible deleterious effects of maternal generations. If early mortality is still observed after careful synchronization, consider using newly prepared culture media, another lot of feeding algae, or a newly established experimental cohort (i.e., start from protocol 3.1).
A limitation of this protocol is that the well-synchronized rotifers are potentially over-sensitive. For example, upon screening of chemicals that extend lifespan, some chemicals screened by this protocol may fail to detect significant effects on lifespans of poorly synchronized rotifers (e.g., individuals from wild and batch-cultured populations). Thus, the results of such experiments should be interpreted with caution.
The effect of maternal age on offspring survival time has also been reported in other invertebrate models including the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans 39,40. Although it is more time-consuming in these long-lived models, the synchronization procedure over several generations would be useful for these animals to decrease experimental variations in measurement of survival time.

Rotifers are microscopic cosmopolitan zooplankton (&#60;1 mm) that constitute the phylum Rotifera 1. They have a simple body plan composed of approximately 1,000 somatic cells as well as a characteristic wheel-like ciliary apparatus called the corona, which is used for locomotion and feeding. Most rotifers belong to classes Monogononta or Bdelloidea, which contain about 1,600 and 500 species, respectively 2. Monogonont rotifers generally have both sexual and asexual reproductive phases (cyclical parthenogenesis), while bdelloid rotifers reproduce by obligatory parthenogenesis 3. It is thus possible to obtain genetically identical rotifer individuals, which ensures high reproducibility in experiments. In addition, they have several other advantages as model organisms, such as a short lifespan, ease of culture, availability of genomic and transcriptomic sequence data 4-7, and a unique phylogenetic position distant from arthropods and nematodes 8. Rotifers are therefore promising invertebrate models in ecological, toxicological, and aging studies 9-12.
The survival time under exposure to environmental stress or chemicals is a frequently measured parameter in these research fields 13-19. However, caution is needed when measuring the survival time of rotifers because it is susceptible to environmental conditions of their mothers. Namely, in the monogonont rotifer Brachionus manjavacas, female offspring from aged mothers have a shorter lifespan than those from young mothers; however, maternal calorie restriction (CR) partially offsets the deleterious effects of advanced maternal age 20. In B. plicatilis, maternal CR provides offspring longevity, long survival time under starvation, and high oxidative stress resistance associated with enhanced expression of antioxidant enzymes 21,22. The maternal age effect has also been observed in bdelloid rotifers 23. Therefore, the conditions of experimental rotifers should be carefully synchronized over several generations before measurements of survival time.
Here we provide a protocol for measurement of survival time in Brachionus rotifers following synchronization of culture conditions over several generations. Intermittent fasting (IF), a variation of CR where rotifers are fed periodically, was applied to reveal the effect of synchronization due to the well-known effects of IF on longevity 22,24.

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measurement of survival time in brachionus rotifers: synchronization of maternal conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Figure 1 shows representative survival curves of poorly synchronized populations (out of two replicates). In this experiment, rotifers were either fed everyday [ad libitum (AL) group] or every other day (IF group). Median survival was 13 and 18 days in the AL and IF groups, respectively. Although it is well known that IF extends the lifespan of the rotifer, this experiment failed to detect a statistically significant difference between lifespans of the AL and IF groups. Empirically, insufficient synchronization results in early mortality and gradual declines in survival rate as observed in this experiment. Damage to rotifers caused by inappropriate treatment or low water quality for the sub-population tends to yield similar results.
When rotifer conditions are optimal and well synchronized, early mortality is hardly observed and accordingly rotifers tend to die in a synchronized way during the later phase of the experiment (Figure 2). Median survival was 13 and 20 days in the AL and IF groups, respectively. Although fewer animals were used than the experiment in Figure 1, the difference in lifespan between these groups was statistically significant. This is the representative results from more than five experiments that have been published previously 22.
<img alt="Figure 1" src="/files/ftp_upload/54126/54126fig1.jpg" /> 
Figure 1: Kaplan-Meier curves for poorly synchronized individuals subjected to intermittent fasting (IF). The AL group was fed ad libitum throughout the experiment, whereas the IF group was fed every other day. N = 11 and N = 12 for the AL and IF groups, respectively (N refers to number of individual used in the experiment). The experiment was performed at 25 &#176;C. No significant difference in lifespan was detected when log-rank test was used (P = 0.1207). However, this data is difficult to interpret because log-rank test should not be used to compare two crossing survival curves although the test is known to be robust 36. No established methods are currently available for crossing survival curves with censored data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54126/54126fig2.jpg" /> 
Figure 2: Kaplan-Meier curves for synchronized individuals subjected to IF. The rotifer cohort, obtained by pre-culture, was subjected to the same IF schedule (fed every other day). N = 6 and N = 8 for AL and IF groups, respectively. The experiment was performed at 25 &#176;C. Log-rank test, P = 0.0057. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions at JoVE.com

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

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8.4K Views.  The University of Tokyo. Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Video: Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

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.

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