We would like to thank Northumbria University for their support.

1. Stock solution preparation
<ol>
	<li>When preparing Calcein stocks solutions, prevent photo degradation by keeping solutions in the dark. Prepare a 1 mM solution of Calcein in Dulbecco&#39;s PBS, without magnesium or calcium14. For 5 mL of 1 mM Calcein solution, add 3.1 mg of Calcein (622.5 g/M) to 5 mL of Dulbecco&#39;s PBS. Using the 1 mM sock of Calcein, prepare 1 mL aliquots of secondary stock solutions in microfuge tube as detailed in Table 1.</li>
	<li>Prepare ferric ammonium sulphate hexahydrate stocks by dissolving 19 mg of FAS hexahydrate (392.1 g/M) in 5 mL of Dulbecco&#39;s PBS to obtain a 10 mM ferric ammonium sulphate (FAS) solution. Prepare secondary stocks of FAS by dilution. To prepare a stock of 2 mM FAS (fA), dilute 200 &#181;L of the 10 mM FAS stock with PBS with 800 &#181;L of Dulbecco&#39;s PBS. To prepare a stock of 100 &#181;M FAS (fB) dilute 50 &#181;L of stock fA with 950 &#181;L of Dulbecco&#39;s PBS.</li>
	<li>To prepare 10 mM of Deferiprone stock, dissolve 2.8 mg (139.15 g/M) in 2 mL of Dulbecco&#39;s PBS. Prepare secondary stocks of 2mM iron chelator by diluting 200 &#181;L of the 10 mM stock with 800 &#181;L of Dulbecco&#39;s PBS. 
	NOTE: In this assay the iron chelator of choice should be dissolved in Dulbecco&#39;s PBS without magnesium or calcium. As a working exemplar, we used the iron chelator Deferiprone.</li>
</ol>
2. Assay validation by determination of the linear range of Calcein fluorescence
<ol>
	<li>Prepare a range of Calcein stocks 1 mM to 1 &#181;M as described in stock solution table (see Table 1). Keep stock solutions in the dark to prevent photochemical degradation.</li>
	<li>Pipette 90 &#181;L of phosphate buffered saline (PBS) to wells A to H and 1 to 12 of a 96 well plate using a single channel manual pipette or a multichannel manual pipette.</li>
	<li>Add 10 &#181;L of 1 mM Calcein stock into column 12, rows A to H, of the 96 well plate using a single channel manual pipette.</li>
	<li>Add 10 &#181;L of the pre-prepared 800 &#181;M Calcein stock (Table 1) to a 96 well plate in column 11, rows A to H, using a single channel manual pipette.</li>
	<li>Repeat the addition procedure with the stocks from Table 1 so that column 10 contains 10 &#181;L of the 400 &#181;M stock and, column 9 contains 10 &#181;L of the 200 &#181;M stock etc. Continue this stepwise procedure across the columns until column 2 contains 10 &#181;L of the 1 &#181;M stock. 
	NOTE: The final concentration of Calcein in each well is 10-fold lower than the stock solutions due to the 1:10 dilution.</li>
	<li>Add 10 &#181;L of PBS to Column 1, row A to E.</li>
	<li>Incubate at room temperature for 30 min and set the analyzer on a microplate reader to an excitation of 490 nm and detection of 530 nm (or suitable filter set for Calcien fluorescence).</li>
</ol>
3. Assay validation by Fe2+ ion quenching to reduce Calcein fluorescence
<ol>
	<li>Prepare a Calcein (cB) stock of 10 &#181;M and a FAS stock (fA) of 2 mM (see Table of Materials). Keep the Calcein stock in the dark to prevent photodegradation.</li>
	<li>Using a single channel manual pipette or a multichannel manual pipette, add 50 &#181;L of PBS to columns 2 to 10, rows A to E. Do not add PBS to column 11.</li>
	<li>Add 100 &#181;L of a FAS (fA) stock of 2 mM into a 96 well plate in column 11 (Rows A to E) using a single channel manual pipette.</li>
	<li>Using a multichannel manual pipette remove 50 &#181;L of fA from column 11 and pipette it into the PBS already in column 10, mix thoroughly by pipetting.</li>
	<li>Continue this stepwise double dilution procedure across the columns 9-2, rows A to E of the 96 well plate, and mix by pipetting.</li>
	<li>Remove 50 &#181;L of solution by pipetting from column 2 and discard the solution to waste.</li>
	<li>Add 40 &#181;L of PBS into all wells with a solution in them, columns 2-12, rows A to E.</li>
	<li>Add 90 &#181;L of PBS to wells column 1, rows A-E. These wells act as a positive control of 1 &#181;M Calcein and 0 &#181;M FAS.</li>
	<li>Add 10 &#181;L of 10 &#181;M Calcein stock (cB) into each well with a solution in it (columns 1 to 12, rows A to E). This results in a top concentration of 1000 &#181;M FAS in column 11, with FAS concentration halving for columns 10, 9, 8 across to column 2. Each of the resultant wells from the columns contains 1 &#181;M Calcein and a dilution of FAS.</li>
	<li>Add 100 &#181;L of PBS to row F of columns 1 to 5 for the PBS alone control. Add 100 &#181;L of 2 mM of FAS to row F of columns 6 to 10 for FAS alone control. Incubate at room temperature for 10 min in the dark.</li>
	<li>Analyze on a microplate reader set to excitation of 490 nm and detection of 530 nm (or suitable filter set for Calcein fluorescence).</li>
</ol>
4. Assay for quantifying the ability of iron chelators to outcompete the weak chelator Calcein for Fe2+ ions
<ol>
	<li>Prepare a Calcein stock (cB) of 10 &#181;M, FAS stock (fB) of 100 &#181;M and the iron chelator stocks of 2 mM as outlined in Table 1. Keep Calcein stock in the dark to prevent photodegradation.</li>
	<li>Using a single channel manual pipette or a multichannel manual pipette, add 50 &#181;L of PBS to columns 2 to 10, rows A to E, of a 96 well plate. Do not add PBS to column 11.</li>
	<li>Add 100 &#181;L of the 2 mM stock (fA) of the iron chelators, into a 96 well plate in column 11, rows A to E using a single channel manual pipette.</li>
	<li>Using a multichannel manual pipette remove 50 &#181;L of fA from column 11 and pipette it into the PBS in column 10, rows A to E, mixing thoroughly by pipetting.</li>
	<li>Continue this stepwise double dilution procedure across the remaining columns 9-2, rows A to E of the plate, mix each well by pipetting. Remove 50 &#181;L from column 2 and discard. This ensures a double dilution of 2 mM of the iron chelator (Deferiprone) across the 96 well plate columns 11 to 2, rows A to E.</li>
	<li>Pipette 30 &#181;L of PBS into all wells with a solution in them (e.g., columns 2-11 rows A to E).</li>
	<li>Using a multi-channel pipette, add 10 &#181;L of 100 &#181;M FAS stock (fB) into columns 2-12, rows A to E. This results in a concentration of 1 &#181;M Calcein and 10 &#181;M FAS in each well, and a double dilution of the chelators, with the concentration halving each column across the plate, columns 11-2, with the highest concentration of iron chelator at 1000 &#181;M in column 11 wells A to E.</li>
	<li>Using a multi-channel pipette add 10 &#181;L of 10 &#181;M Calcein stock (cB) into columns 2-12, rows A to E.</li>
	<li>Pipette 80 &#181;L of PBS and 10 &#181;L of Calcein stock (cB) and 10 &#181;L of FAS stock (fB) to column 1, rows A-E using a pipette. Mix samples by pipetting. This provides the control of 1 &#181;M Calcein with 10 &#181;M FAS and no chelator.</li>
	<li>Pipette 100 &#181;L of PBS into row F columns 1 to 5. This provides the PBS negative control.</li>
	<li>Add 100 &#181;L of 2 mM of iron chelator (Deferiprone) to row F, columns 6 to 10. This provides the chelator alone control.</li>
	<li>Incubate at room temperature for 10 min in the dark. Analyze on a multimodal microplate reader set to excitation at 490 nm and detection at 530 nm (or suitable filter set for Calcien fluorescence).</li>
</ol>
5. Data analysis
<ol>
	<li>Analysis for assay validation by determination of the linear range of Calcein fluorescence.
	<ol>
		<li>Calculate the mean Relative Fluorescence Units (RFU) of rows A-H for each concentration of Calcein in the columns of the 96 well plate from step 1 and plot against the concentration. Plot the line graph of Calcein concentration on the x-axis against mean RFU on the y-axis and use a linear regression to provide line of best fit.</li>
		<li>Quantify statistical significance by comparing Calcein concentrations in each column (2-11) to the control of PBS in column 1 rows A to E. Perform an Analysis of variance (ANOVA) and apply post hoc analysis using Least Significant Difference (LSD) with a threshold of p &#60;0.001 to determine statistical significance, (denoted by ** in Figure 1).</li>
	</ol>
	</li>
	<li>Analysis for assay validation by Fe2+ ion quenching to reduce Calcein fluorescence.
	<ol>
		<li>Calculate the average Relative Fluorescence Units (RFU) of rows A to E for each concentration of FAS in columns 2-11. Plot mean RFU for each FAS concentration on the y-axis. On the x-axis plot concentrations of FAS using a Log2&#160;scale as itbetter distributes the data. Plot a line of best fit using a linear regression.</li>
		<li>Quantify statistical significance by comparing each columns mean to mean RFU of the control of PBS, 1 &#181;M Calcein, 0 &#181;M FAS (column 1 row A to E). Perform an ANOVA with LSD post hoc analysis with a threshold of p &#60;0.001 to determine statistical significance.</li>
	</ol>
	</li>
	<li>Analysis for assay: Quantifying the ability of iron chelators to outcompete the weak chelator Calcein for Fe2+ ions.
	<ol>
		<li>Calculate the average Relative Fluorescence Units (RFU) of rows A to E for each concentration of chelator in columns 2-11. Plot mean RFU for each chelator concentration on the y axis. On the x-axis plot concentrations of chelator using a Log2 scale as it better distributes the data.</li>
		<li>Quantify statistical significance by comparing each columns mean to mean RFU of the control of PBS 1 &#181;M Calcein and 10 &#181;M FAS (column 1 row A to E). Perform an ANOVA with LSD post hoc analysis with a threshold of p &#60;0.001 to determine statistical significance.</li>
	</ol>
	</li>
</ol>

Fluorescent Assay for Detection of Novel Iron Chelators

<ol>
	<li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Hsu, M. Y., Mina, E., Roetto, A., Porporato, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron:+An+essential+element+of+cancer+metabolism.">Iron: An essential element of cancer metabolism.</a> Cells. 9 (12), 2591 (2020).</li><li>Torti, S. V., Torti, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer:+More+ore+to+be+mined.">Iron and cancer: More ore to be mined.</a> Nat Rev Cancer. 13 (5), 342-355 (2013).</li><li>Zhang, C., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+homeostasis+and+tumorigenesis:+Molecular+mechanisms+and+therapeutic+opportunities.">Iron homeostasis and tumorigenesis: Molecular mechanisms and therapeutic opportunities.</a> Protein Cell. 6 (2), 88-100 (2015).</li><li>Torti, S. V., Manz, D. H., Paul, B. T., Blanchette-Farra, N., Torti, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer.">Iron and cancer.</a> Annu Rev Nutr. 38, 97-125 (2018).</li><li>Welch, D. R., Hurst, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+hallmarks+of+metastasis.">Defining the hallmarks of metastasis.</a> Cancer Res. 79 (12), 3011-3027 (2019).</li><li>Lehmann, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+defects+of+hepatocellular+carcinoma+are+already+found+in+non-neoplastic+liver+cells+from+patients+with+hereditary+haemochromatosis.">Epigenetic defects of hepatocellular carcinoma are already found in non-neoplastic liver cells from patients with hereditary haemochromatosis.</a> Hum Mol Genet. 16 (11), 1335-1342 (2007).</li><li>Fonseca-Nunes, A., Jakszyn, P., Agudo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer+risk--a+systematic+review+and+meta-analysis+of+the+epidemiological+evidence.">Iron and cancer risk--a systematic review and meta-analysis of the epidemiological evidence.</a> Cancer Epidemiol Biomarkers Prev. 23 (1), 12-31 (2014).</li><li>Abdelaal, G., Veuger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+oncogenic+transformation+with+iron+chelation.">Reversing oncogenic transformation with iron chelation.</a> Oncotarget. 12 (2), 106-124 (2021).</li><li>Brown, R. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+iron+metabolism+and+impact+in+cancer+biology,+metastasis,+and+immunology.">Altered iron metabolism and impact in cancer biology, metastasis, and immunology.</a> Front Oncol. 10, 476 (2020).</li><li>Menyh&#225;rt, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+selection+of+functional+assays+to+evaluate+the+hallmarks+of+cancer.">Guidelines for the selection of functional assays to evaluate the hallmarks of cancer.</a> Biochim Biophys Acta. 1866 (2), 300-319 (2016).</li><li>Basu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+novel+and+diverse+iron-chelators+in+silico.">Discovering novel and diverse iron-chelators in silico.</a> J Chem Inf Model. 56 (12), 2476-2485 (2016).</li><li>Prus, E., Fibach, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+measurement+of+the+labile+iron+pool+in+human+hematopoietic+cells.">Flow cytometry measurement of the labile iron pool in human hematopoietic cells.</a> Cytometry A. 73 (1), 22-27 (2008).</li><li>Cold Spring Harb. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Phosphate-buffered saline (pbs). 2006 (1), (2006).</li><li>Kontoghiorghes, G. 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The authors declare no conflict of interest.

The over reliance of cancers on iron to fuel their metabolism makes iron chelation a potential addition to therapeutic regimes4. However, there is a limited ability to quickly screen novel metal ion chelators for their ability to bind iron ions. The commonly used and widely available fluorescent probe Calcein is known to act as a weak iron chelator and binding by iron ions quenches Calcein fluorescence. Fluorescence can then be recovered by competing for binding to iron using a stronger iron chela...

Phenotypic changes to cells that relate to the development of cancer through a common set of altered biological capabilities are now commonly referred to as the hallmarks of cancer. Amongst them are changes resulting from the reprogramming of energy metabolism, which are widespread in cancer cell biology1. Such metabolic reprogramming includes an increased requirement for iron to support rapid proliferation and tumor growth2. This thirst for iron leads to dysregulated iron metabolism, which in and of itself is considered a hallmark of cancer3,4, with dysregulation occurring at all stages5. The hallmarks of metastasis, more recently proposed by Welch and Hurst, include a role for iron6 since iron can induce oxidative stress, and this can, in turn, mediate changes to the genome, epigenome, and proteome, enhancing the possibility of metastasis7. A link between iron levels and an increased occurrence of cancer has been demonstrated through epidemiological studies8.
Since cancer cells require large amounts of iron, they are susceptible to iron deficiency and, therefore, iron chelation. We have recently published a review article highlighting the potential for iron chelation in reversing several hallmarks of cancer through NDRG1 disrupting oncogenic signaling pathways9. However, the use of iron chelation as stand-alone cancer therapy has not yielded positive results in clinical trials due to their toxicity, short half-life, rapid metabolism, and emerging resistance mechanisms. Nevertheless, iron chelators have shown promise in in vitro and in vivo investigations, indicating that more work is needed to develop effective iron chelators for cancer therapy. Specific iron chelation is a validated strategy in anticancer drug discovery, but only a few classes have been reported to date10.
The identification and characterization of novel iron chelators requires the ability to measure their effect on several endpoints. Many of these (such as proliferation, apoptosis, reactive oxygen species formation) are routinely measured and have been outlined and reviewed in the literature as methods to evaluate the hallmarks of cancer11. When evaluating a novel iron chelator, many groups routinely examine the effect on anti-proliferative and redox activities as well as effects on iron influx or efflux. In silico prediction techniques12 further increase the growing pool of iron chelators that can be screened.
Screening of iron chelators requires the ability to measure their effect on iron levels as a means to effectively demonstrate proof of principle. Currently, the most common method to do so is flow cytometry13 which is costly, time consuming and poorly quantifiable. The choice of assay is often based on the availability of experimental equipment, speed, and cost of an assay. Therefore, the ability to chelate iron can be an 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.

<table><tbody><tr><td>Ammonium iron(II) sulfate hexahydrate</td><td>Sigma-Aldrich</td><td>215406</td><td>other wise known as FAS</td></tr><tr><td>Calcein</td><td>Sigma-Aldrich</td><td>C0875</td><td></td></tr><tr><td>Deferiprone</td><td>Sigma-Aldrich</td><td>379409</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D5652</td><td>magnesium and calcium free</td></tr><tr><td>Greiner CELLSTAR 96 well plates</td><td>Sigma-Aldrich</td><td>M0812</td><td>any optically transparent 96 well plate will work</td></tr></tbody></table>

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.

For the method shown in step 2, this first experiment (Figure 1) established the linear range of the microtiter fluorescence plate reader when detecting Calcein fluorescent emissions. Our representative results show a wide linear range of Calcein fluorescence from 0-100 &#181;M. ANOVA with LSD post hoc analysis demonstrates that there is a statistically significant differences in mean RFU for all Calcein concentrations when compared to a control of 0 &#181;M of Calcein. In further experiment...

Read this Scientific Article Protocol about Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron at JoVE.com

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.

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 ...

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Research

JoVE Journal

Cancer Research

361 Views.  Northumbria University. 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. 

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

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

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.

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 ...

Biochemistry

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

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Bioengineering

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

Biology

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 ...

Ferritinophagy: Assessing the Selective Degradation of Iron by Autophagy in Human Fibroblasts

Mutations in the autophagy gene WDR45/WIPI4 are the cause of beta-propeller-associated neurodegeneration (BPAN), a subtype of human diseases known as neurodegeneration with brain iron accumulation (NBIA) due to the presence of iron deposits in the brains of patients. Intracellular iron levels are tightly regulated by a number of cellular mechanisms, including the critical mechanism of ferritinophagy. This paper describes how ferritinophagy can be assessed in primary, skin-derived human fibroblasts. In this protocol, we use iron-modulating conditions for inducing or inhibiting ferritinophagy at the cellular level, such as the administration of bafilomycin A1 to inhibit lysosome function and ferric ammonium citrate (FAC) or deferasiox (DFX) treatments to overload or deplete iron, respectively. Such treated fibroblasts are then subjected to high-throughput imaging and CellProfiler-based quantitative localization analysis of endogenous ferritin and autophagosomal/lysosomal markers, here LAMP2. Based on the level of autophagosomal/lysosomal ferritin, conclusions can be drawn regarding the level of ferritinophagy. This protocol can be used to assess ferritinophagy in BPAN patient-derived primary fibroblasts or other types of mammalian cells.

This protocol provides detailed instructions for performing high-throughput, immunofluorescence-based ferritinophagy assessments in primary, skin-derived human fibroblasts.

Mutations in the autophagy gene WDR45/WIPI4 are the cause of beta-propeller-associated neurodegeneration (BPAN), a subtype of human diseases known as ...

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)
...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

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 ...

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

Summary

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Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

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

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

Ferritinophagy: Assessing the Selective Degradation of Iron by Autophagy in Human Fibroblasts

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

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