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 &amp;mu;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>&lt;em&gt;Tobacco, Nicotiana tabacum&lt;/em&gt; 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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Video: A Colorimetric Method for Measuring Iron Content in Plants

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

Environment

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III))

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative conditions. Excessive iron results in free redox-active Fe(II) and can cause devastating effects within the cell like oxidative stress (OS) and death by lipid peroxidation known as ferroptosis (FPT). Therefore, quantitative measurements of ferrous (Fe(II)) and ferric (Fe(III)) iron rather than total Fe-determination is the key for closer insight into these detrimental processes. Since Fe(II)/(III) determinations can be hampered by fast redox-state shifts and low concentrations in relevant samples, like cerebrospinal fluid (CSF), methods should be available that analyze quickly and provide low limits of quantification (LOQ). Capillary electrophoresis (CE) offers the advantage of fast Fe(II)/Fe(III) separation and works without a stationary phase, which could interfere with the redox balance or cause analyte sticking. CE combined with inductively coupled plasma mass spectrometry (ICP-MS) as a detector offers further improvement of detection sensitivity and selectivity. The presented method uses 20 mM HCl as a background electrolyte and a voltage of +25 kV. Peak shapes and concentration detection limits are improved by conductivity-pH-stacking. For reduction of 56[ArO]+, ICP-MS was operated in the dynamic reaction cell (DRC) mode with NH3 as a reaction gas. The method achieves a limit of detection (LOD) of 3 &#181;g/L. Due to stacking, higher injection volumes were possible without hampering separation but improving LOD. Calibrations related to peak area were linear up to 150 &#181;g/L. Measurement precision was 2.2% (Fe(III)) to 3.5% (Fe(II)). Migration time precision was &#60;3% for both species, determined in 1:2 diluted lysates of human neuroblastoma (SH-SY5Y) cells. Recovery experiments with standard addition revealed accuracy of 97% Fe(III) and 105 % Fe(II). In real-life bio-samples like CSF, migration time can vary according to varying conductivity (i.e., salinity). Thus, peak identification is confirmed by standard addition.

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII

This iron redox speciation method is based on capillary electrophoresis-inductively coupled plasma mass spectrometry with sample stacking combined with short analysis in one run. The method quickly analyzes and provides low limits of quantification for iron redox species across a diverse range of tissues and biofluid samples.

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative ...

Chemistry

Fluorescent Assay for Detection of Novel Iron Chelators

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Author Spotlight: Assessing the Impact of Novel Iron Chelators on Cancer Cell Metabolism

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

Cancer Research

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

Medicine

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal iron endowment is entirely dependent on iron transfer across the placenta, and perturbations of this transfer can lead to adverse pregnancy outcomes. In mice, measurement of iron fluxes across the placenta traditionally relied on radioactive iron isotopes, a highly sensitive but burdensome approach. Stable iron isotopes (57Fe and 58Fe) offer a nonradioactive alternative for use in human pregnancy studies.
Under physiological conditions, transferrin-bound iron is the predominant form of iron taken up by the placenta. Thus, 58Fe-transferrin was prepared and injected intravenously in pregnant dams to directly assess placental iron transport and bypass maternal intestinal iron absorption as a confounding variable. Isotopic iron was quantitated in the placenta and mouse embryonic tissues by inductively coupled plasma mass spectrometry (ICP-MS). These methods can also be employed in other animal model systems of physiology or disease to quantify in vivo iron dynamics.

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described.

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal ...

Biology

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

Introduction to Mass Spectrometry

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.
A mass spectrometer is composed of an ionization source, an analyzer,&#160;and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.
In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.

Mass Spectrometer: Principle, Operation, Applications

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the ...

Education

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Analytical Chemistry

Bathophenanthroline Sulfonate-Based Colorimetric Assay: A Simple and Rapid Method for Quantitation of Non-Heme Iron in Mouse Liver Tissue

Source: Duarte, T. L., Neves, J. V. <a href="https://www.jove.com/v/63469">Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay.</a> J. Vis. Exp. (2022)
In this video, we describe a colorimetric-based method to determine non-heme iron content in mouse liver tissue. Bathophenanthroline sulfonate is used as the chromogenic substrate in the presence of a reducing agent, which converts ferric iron to chelatable ferrous iron.

Source: Duarte, T. L., Neves, J. V. Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay. J. Vis. Exp. (2022)
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Biological Techniques

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Biochemical assays

Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources

Source: Glahn, R. P. <a href="https://app.jove.com/v/63859">The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability.</a> J. Vis. Exp. (2022)
This video demonstrates an in vitro technique to assess iron bioavailability in complex food sources. The technique combines the simulation of human digestion along with iron uptake by differentiated Caco-2 cells. Intracellular ferritin formation acts as a marker for cellular iron uptake.

Source: Glahn, R. P. The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability. J. Vis. Exp. (2022)
This video demonstrates an in vitro ...

A Colorimetric Method for Measuring Iron Content in Plants

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