This work was supported by the Israel Ministry of Science, Technology and Spaceand by a grant from the Chief Scientist of the Israeli Ministry of Agriculture (#16-16-0003).

1. Plant Material and Growth Conditions
<ol>
	<li>Seed one tobacco (cultivar Samsun) seed per 5 cm x 5 cm pot filled with standard pot medium. Place the pots on trays. Grow the plants in a growth room under long day conditions (16/8 h light/dark) at a constant temperature of 23 &#176;C. Irrigate with tap water until water drains from the pot.</li>
	<li>After 50&#177;5 days, start Fe treatments in the irrigation, according to the concentrations suitable for the experiment. For example, we used a range of iron concentrations from 0 to 6 mM, supplemented by a soluble Fe chelator (Fe EDDHA). Irrigate the plants with the appropriate solution every two days (to avoid dehydration) for 6-8 days.</li>
</ol>
2. Preparing the Leaves for Iron Measurement
Notes: All materials to be used must be iron-free so as to reduce the risk of iron contamination. Clean the mortar and pestle twice with 4% HCl solution and dry with filter paper each time before use. If any material is reused, clean it twice with 4% HCl solution and dry with filter paper.
<ol>
	<li>Detach the leaves from the stem by hand, using gloves (do not use any metal equipment). Use about 10 g of leaves (fresh weight) for each sample. Clean each leaf with double distilled water (DDW) using a spray bottle. This step is important to avoid Fe contamination.</li>
	<li>Dry the leaves on a paper towel and put them in a paper bag. Transfer the paper bags to an oven at a constant temperature of 80 &#176;C for 2-3 days.</li>
	<li>When dry, crush the leaves to powder using a mortar and pestle and transfer to sterile 15 mL plastic tubes.</li>
</ol>
3. Burning the Leaves to Ash
Notes: The use of a low pH (close to 0) solution of HCl is meant to increase iron solubility. The rock wool is used to prevent the gases from escaping the vial during burning.
<ol>
	<li>Weigh a new, sealed 20 mL scintillation vial without its lid. Note the value or set the value to zero using the tare button. Add the crushed dried leaves (sample) to the vial.</li>
	<li>Weigh the sample and container and note the value. Close the vial with rock wool.</li>
	<li>Weigh 3 additional vials without adding samples and note their values. These vials will be used as controls to evaluate the amount of rock wool that could have led to any increase in sample weight.</li>
	<li>Place the sample and control vials in a furnace and start burning using the following temperature steps: room temperature, fast increase to 425 &#176;C, and, finally, 425&#176;C for 4 hours. By this time, the dry leaves will have turned to ash.</li>
	<li>Let the samples cool down to about 100 &#176;C but not below this temperature for the following two steps to avoid humidity, which could affect the final weight of the sample. Using heavy gloves, remove the samples from the furnace with tweezers, holding the vial exteriorly.</li>
	<li>Place the vials on a flat surface, remove the rock wool and close the vials with their original lids.</li>
	<li>Weigh the 3 control vials (see 3.3) and calculate their average weight gain. If weight gain is equal or above to 1% of the ash weight (see step 4.2), use this value as an estimate of the measurement error.</li>
</ol>
4. Preparing the Ashes for Iron Measurement
Notes: The final iron concentration in the initial sample is calculated as the weight of the ashes divided by the added volume of HCl.
<ol>
	<li>Prepare a 1 M HCl solution (4% HCl) by adding 12.5 mL of a 37% HCl stock solution to 87.5 mL of DDW (in a plastic or glass flask).</li>
	<li>Weigh a 15 mL plastic tube and note the value or set the value to zero using the tare button. Transfer the ashes to the tube, weigh, and note the value. This is the ash weight.</li>
	<li>Add 5 mL of 1 M HCl to the ashes. Filter the ashes through a 22 &#181;m filter and add an additional 5 mL of 1 M HCl through the same filter.</li>
	<li>The final volume should be 10 mL. Note that part of the solution will be lost in the filter. 
	NOTE: The samples are now ready for Fe measurement either by FAAS or by the Prussian Blue method.</li>
	<li>Make a calibration curve with the Fe concentration measured by atomic spectrometry and by the Prussian blue method (see Figure 4) for each plant species. Subsequently, Fe concentration can be measured by the Prussian blue method alone.</li>
</ol>
5. Measuring Fe Concentration by FAAS
<ol>
	<li>Remove 4 mL from each sample for measurement by FAAS.</li>
	<li>Divide the results obtained from the FAAS measurement by the weight of the ashes. Divide the resulting value by 0.01 (because the ashes were solubilized in 10 mL). The resulting value is the iron concentration per gram ash (ppm).</li>
</ol>
6. Preparing the Prussian Blue Staining Solution
<ol>
	<li>Prepare a 4% Prussian blue solution by adding 4 g of K4Fe(CN)6 to 100 mL DDW and vortex (other volumes and/or concentrations can be used for different demands). It should be noted that in this study, a less concentrated Prussian blue solution than previously reported (20%)14 was used.</li>
	<li>Keep the solution in the dark at 4 &#176;C until use. The solution is stable for 6 months when stored at such conditions.</li>
</ol>
7. Generating a Calibration Curve for the Prussian Blue Method Using FAAS Results
NOTE: Calculate the iron concentration in the ashes using the following formula 
<img alt="Equation" src="/files/ftp_upload/57408/57408eq1.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 1 
C: concentration, V: sample volume, W: ash weight (g).
<ol>
	<li>Mix 0.50 mL of Prussian blue solution and 0.50 mL of 1 M HCl. This will serve as the blank solution.</li>
	<li>Mix 0.5 mL of sample (ashes in 4% HCl, as described in section 3) and 0.5 mL of Prussian blue solution (step 6.1) by pipetting. Wait at least 1 minute but not more than 5 minutes. After 5 minutes, sedimentation in the samples will occur.</li>
	<li>Transfer the mix to a cuvette and measure the OD at 715 nm using a spectrophotometer. Note the value.</li>
	<li>Divide the OD value (step 7.3) by the ash weight (step 3.2) of the sample. The result represents OD per gram ash.</li>
	<li>Plot the linear regression between the iron concentrations obtained from the FAAS measurements (Y axis) and the OD values (X axis). Use the results obtained in steps 5.2 and 7.4. Calculate the regression formula, Y = a + bX, where Y represents iron concentration, a represents the absorbance intersect, b represents the absorbance slope and X represents the OD.</li>
</ol>
8. Using the Prussian Blue Method for Determining Iron Levels in Other Samples from the Same Plant Type
Notes: Since a calibration curve has already been established for this type of plant, iron concentration in any new samples from the same plant type can be directly calculated using the linear regression formula.
<ol>
	<li>Perform the steps in sections 3 and 4, followed by steps 7.1 to 7.4.</li>
	<li>Calculate the iron concentration in solution using the formula obtained from the linear regression (step 7.5).</li>
</ol>

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The authors have nothing to disclose.

Iron measurement in plant tissues is very important for evaluating the effects of irrigation or other environmental conditions. Here, we described an easy and accurate colorimetric method for Fe content measurement in tobacco leaves, which can be readily adapted to other plant species and tissues.
In optimizing conditions for the colorimetric method, we used a low pH medium (pH &#60; 1.0) to allow iron solubility. The burning process was performed to release all forms of iron and to ensure tha...

Iron (Fe) is an important micronutrient in all living organisms. In plants, it is an essential micronutrient1 because of its involvement in basic processes, such as respiration, photosynthesis and chlorophyll biosynthesis. High accumulation of free iron ions is harmful to plant cells due to reactions leading to the release of free radicals causing oxidative stress. To maintain iron homeostasis within the plant cell, ions are stored in vacuoles and sequestered within ferritins, protein cages directly involved in iron homeostasis2 and the principal storage structure of iron in all living organisms. At the same time, iron-deficiency anemia affects a significant proportion of the human population, resulting in an increasing need for plant Fe biofortification. Due to the unique properties of plant ferritin, food enrichment with ferritin-iron offers a promising strategy to fight this problem of malnutrition3.
Iron ions are mainly found in two oxidation states, namely the ferrous (divalent Fe2+ or iron (II)) and ferric (trivalent Fe3+ or iron (III)) forms. Several other forms of iron, such as iron clusters4, are also found in cells. Fe is stored as iron oxide within the cell and naturally forms hematites (Fe2O3) and ferryhidrites ((Fe3+)2O3&#8226;0.5H2O) under physiological conditions5. The hydroxides formed in these reactions, especially the ferric form, have very low solubility. Iron retention is consequently affected by the pH of the solution and is largely in a solid state above pH 56.
Considering the poor solubility and high reactivity of Fe, its transfer among plant tissues and organs must be associated with suitable chelating molecules. Moreover, its redox states between the ferrous and ferric forms1 must be controlled. Within leaves, about 80% of the iron is found in photosynthetic cells, due to its essential roles in the electron transport system, in the biosynthesis of cytochromes, chlorophyll and other heme molecules, and in the formation of Fe-S clusters7. In the case of iron excess within the cell, the surplus is translocated into the vacuole where the metal is stored in ferritin molecules8.
Iron can be measured in plant tissues by several methods, including flame atomic absorption spectroscopy9 (FAAS) or colorimetric assays10, the former being far more precise than the latter. FAAS is a highly accurate technique that enables one to determine the elemental composition of a sample on the basis of the electromagnetic emission of the individual elements. FAAS converts metal ions to atomic states by flame-heating of the sample, leading to ion excitation and emission of a specific wavelength when a given ion returns to its ground state. The emissions from the different ions are separated by a monochromator and detected by an absorption sensor11. FAAS thus serves to directly quantify iron concentrations. Other techniques for visualizing iron in biological tissues are, however, available. Inductively-coupled plasma-mass spectroscopy (ICP-MS)12 is a very precise technique for measuring iron and other trace elements but the lack of equipment, both for FAAS and ICP-MS, is a common problem. On the other hand, iron measurement by thiocyanate colorimetry13 lacks precision and fails to detect small variations between samples. Prussian blue staining14,15,16,17 is an indirect method based on the reaction of potassium ferric ferrocyanide (K4Fe(CN)6) with Fe cations, producing a strong blue color, and is used for qualitative iron detection in histological sections of animal and plant tissues.
Metallic (zero-valent) iron is rare in the lithosphere. The dominant non-complexed ionic form of iron in the environment is mostly dictated by the amount of oxygen in the surroundings, with ferrous iron being relatively more abundant in anoxic environments and ferric iron predominating in aerobic sites. This latter form is also dominant in extremely acidic environments, although the causative agents of ferrous iron oxidation often differ in anoxic and acidic surroundings18. When iron is solubilized in 4% HCl (pH 0) in an aerobic environment, the major part of the diluted iron exists as the ferric form (Fe3+)19,20.
The reactions between Fe ions and K4Fe(CN)6 are as follows:
Fe3+: FeCl3 + K4Fe(CN)6 = KFe(III)Fe(II)(CN)6&#175; + 3KCl
Fe2+: 4 FeCl2 + 2 K4Fe(CN)6 = Fe4(Fe(CN)6)2 + 8 KCl
In the present study, we asked whether Prussian blue staining can be useful for measuring iron levels in solution.
Initially, we verified the correlation between the concentration of Fe in aqueous solution and Prussian blue staining. The Fe (as FeCl2, FeCl3 or a 1:1 mixture of the two) concentration in aqueous solutions was measured both by atomic spectroscopy and by absorbance (OD) after addition of Prussian blue. Figure 1 shows the linear regression curves for measurements obtained by each method. We concluded that the Prussian blue method can be used for quantitative analysis of iron concentration in solution.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57408/57408fig1.jpg" /> 
Figure 1: Linear regressions between Fe concentration measured by FAAS and light absorbance (OD, 715 nm) obtained by the Prussian blue method. The blue squares and line represent the Fe2+ solution, the red squares and line represent the Fe3+ solution and the black squares and line represent a 1:1 mixture between Fe2+ and Fe3+. The following regressions were obtained: [Fe2+] = 3 + 123 x OD, r = 0.996, R2 = 0.989; [Fe3+] = 1 + 292 x OD, r = 0.999, R2 = 0.997; and [Fe2+/3+] = 11 + 146 x OD, r = 0.983, R2 = 0.956. The Fe2+ donor was FeCl2 and the Fe3+ donor was FeCl3. <a href="https://www.jove.com/files/ftp_upload/57408/57408fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To adapt the colorimetric Prussian blue method for quantitative iron analysis of plant tissues, the iron content of tobacco leaf ashes was measured by flame absorption atomic spectroscopy and Prussian blue staining. There was good correlation between the results from by the two techniques.

<table>
	<tbody>
		<tr>
			<td>Potassium Hexacyanoferrate(II)</td>
			<td>Fisher Chemical</td>
			<td>14459-95-1</td>
			<td>Reagent for the Pussian Blue</td>
		</tr>
		<tr>
			<td>Millex Syringe Filter Unit, Vial Vent 0.22 &#956;m</td>
			<td>Millec</td>
			<td>SLGP033RS</td>
			<td>Filter used to filter the ashes + 4% HCl Solution</td>
		</tr>
		<tr>
			<td>Scintillation Vials</td>
			<td>Fisherbrand</td>
			<td>03-337-4</td>
			<td>Used to keep the dry powdered plant material during the burning procedure.</td>
		</tr>
		<tr>
			<td>Disposable Syringe 10 ml</td>
			<td>Medi-Plus</td>
			<td>1931</td>
			<td>Syringe used during the filtration</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>231-595-7</td>
			<td>Used in the 4% HCl solution to dilute the ashes and clean the materials</td>
		</tr>
		<tr>
			<td>Tobacco, Nicotiana tabacum cv. Samsun NN</td>
			<td>Obtained from Prof. Simon Barak and routinely used in the Zaccai Lab</td>
			<td>Barak S, Nejidat A, Heimer Y, Volokita M. Transcriptional and posttranscriptional regulation of the glycolate oxidase gene in tobacco seedlings. Plant Molecular Biology. 2001 Mar 1;45(4):399-407.</td>
			<td>Tobacco cultivar used in this protocol</td>
		</tr>
		<tr>
			<td>Glass Wool (Rock Wool)</td>
			<td>Sigma-Aldrich</td>
			<td>659997-17-3</td>
			<td>Used in the procedure of burning samples in the furnace.</td>
		</tr>
	</tbody>
</table>

a colorimetric method for measuring iron content in plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

When this protocol is carried out correctly, one should get excellent correlation between the results obtained by the Prussian blue and atomic spectroscopy methods. Therefore, the Prussian blue method can be easily used to obtain an accurate measurement of iron concentration in plant samples, as reflected in the following experiment.
Tobacco plants were grown as described in the protocol and irrigated with water containing diffe...

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A Colorimetric Method for Measuring Iron Content in Plants

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

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21.9K Views.  Ben-Gurion University of the Negev. We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Video: A Colorimetric Method for Measuring Iron Content in Plants

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.