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Iron assay kit

Manufactured by Merck Group
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Sourced in United States, China, Germany

The Iron Assay Kit is a laboratory equipment product designed to quantify the concentration of iron in a given sample. It provides a reliable and accurate method for measuring iron levels across various applications.

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Close spelling variants of this product

Iron Colorimetric Assay Kits Prussian blue iron assay kit MAK025 iron assay kit

141 protocols using iron assay kit

1

Retinal Iron Quantification and Localization

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Serum and retinal iron levels (total iron, ferrous iron, and ferric iron) were measured using an Iron Assay Kit (MAK025; Sigma) according to the manufacturer’s instructions. Perl’s staining was used to detect the ferric iron distribution in each layer of the retina. Briefly, deparaffinized and rehydrated retinal paraffin sections (3 µm) were stained using a Prussian blue staining kit (G1029; Servicebio) according to the recommended protocol. FerroOrange (F374; Dojindo, Beijing, China) was applied to reveal the distribution and expression of ferrous iron in the cytoplasm of living R28 cells. Briefly, the R28 cell culture medium was replaced with serum-free medium containing 1 μM FerroOrange, and then the R28 cells were incubated at 37 °C with an atmosphere containing 5% CO2 for 30 min before being detected using a confocal fluorescence microscope (Leica, Frankfurt, Germany).
Yao F., Peng J., Zhang E., Ji D., Gao Z., Tang Y., Yao X, & Xia X. (2022). Pathologically high intraocular pressure disturbs normal iron homeostasis and leads to retinal ganglion cell ferroptosis in glaucoma. Cell Death and Differentiation, 30(1), 69-81.
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2

Acid-Dissolution Assay for Hydroxyapatite Beads

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In this acid-dissolution assay, 10 mg of sHA beads were incubated in 1 ml of 0.1 M NaOAc buffer (pH 4.5) containing 0.5 mg ml-1 CAT-NP for 2 h with rocking at room temperature. Then, the supernatant was removed and sHA beads were resuspended again in fresh 1 ml of acidic NaOAc buffer and incubated as described above. The same procedure was conducted with sHA beads without CAT-NP (control) as well as with sHA beads (with or without CAT-NP) in 0.1 M NaOAc buffer at pH 7.0. An aliquot of sHA immediately before and after acid-dissolution was taken and analyzed via optical microscopy (OM) and SEM. In parallel, the remaining sHA beads were oven-dried and weighed for determination of dry-weight. The remaining dry-weight of sHA treated with CAT-NP was compared to control group to evaluate the efficiency of reduction of demineralization. For iron release assay, 0.5 mg ml-1 CAT-NP was incubated in 0.1 M NaOAc (pH 4.5) at room temperature for 0, 3, 5, 10, 30, 60, 120 min, respectively. The mixture was centrifuged and the supernatant was collected for iron concentration measurement using Iron Assay Kit (Sigma-Aldrich) or ICP-OES. The iron release in 0.1 M NaOAc (pH 7) was conducted as control following the same procedure described above.
Gao L., Liu Y., Kim D., Li Y., Hwang G., Naha P.C., Cormode D.P, & Koo H. (2016). Nanocatalysts promote Streptococcus mutans biofilm matrix degradation and enhance bacterial killing to suppress dental caries in vivo. Biomaterials, 101, 272-284.
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3

Iron Quantification Protocol

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Iron dosage was performed with the Iron Assay kit (Sigma Aldrich) according to the manufacturer’s instructions.
Legendre C., Avril S., Guillet C, & Garcion E. (2016). Low oxygen tension reverses antineoplastic effect of iron chelator deferasirox in human glioblastoma cells. BMC Cancer, 16, 51.
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4

Quantifying Ferrous Iron in HA-TiO2-IONPs

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Iron Assay Kit (Sigma-Aldrich, MO, USA) was used to determine Fe2+ concentration. Dissolve HA-TiO2-IONPs in water with different acidity (pH = 5.5 or 7.4) and GSH content to obtain solutions with the same carrier concentration (100 μg/ml). These samples were labeled as ① (pH = 5.5 without GSH), ② (pH = 7.4 without GSH) and ③ (pH = 5.5 with 5 mM GSH). Shake them on a horizontal shaker (100 rpm) at room temperature. At 4 and 8h, 50 μl sample was drawn and transferred to 96-well plates. Bring samples to a final volume of 100 μl with iron assay buffer. To measure ferrous iron, add 5 μl of iron assay buffer to each well. Mix well and incubate the reaction for 30 min at room temperature in the dark. Add 100 μl of iron probe to each well containing test samples. Mix well and incubate for 60 min without light. At last, measure the absorbance at 593 nm.
Zhang H., Zhang H., Zhu X., Zhang X., Chen Q., Chen J., Hou L, & Zhang Z. (2017). Visible-light-sensitive titanium dioxide nanoplatform for tumor-responsive Fe2+ liberating and artemisinin delivery. Oncotarget, 8(35), 58738-58753.
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5

Quantifying Total and Reduced Iron Levels

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Iron assay kit (MAK025, Sigma, USA) was used directly to detect both total and/or reduced iron concentrations in the samples after addition of acidic buffer. The released iron is reacted with a chromagen resulting in a colorimetric (593 nm) product, the intensity of which is proportional to the iron presented in the cells. After centrifugation at 16,000 × g for 10 min at 4 ℃, the supernatant was discharged. To measure total iron, 50 μL samples were supplemented with 5 μL iron reducer to reduce Fe3+ to Fe2+ and then adjusted to a final volume of 100 μL per well in a 96-well plate with assay buffer. Following a mixture on a horizontal shaker and incubation for 30 min at 25 ℃, 100 μL iron probe was respectively added to each well containing standard and test samples. Thereafter, the samples in the 96-well plate were mixed again and incubated for another 60 min at 25 ℃ in the dark. Finally, the absorbance was measured at 593 nm using a Spectra Max M3 Fluorescence Microplate Reader (Molecular Devices, USA).
Wen J., Chen H., Ren Z., Zhang P., Chen J, & Jiang S. (2021). Ultrasmall iron oxide nanoparticles induced ferroptosis via Beclin1/ATG5-dependent autophagy pathway. Nano Convergence, 8, 10.
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6

Measuring Iron Oxide in LNs

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Iron oxide content in LNs was measured using the Iron Assay Kit (Sigma Aldrich) according to package instructions.
Grippin A.J., Wummer B., Wildes T., Dyson K., Trivedi V., Yang C., Sebastian M., Mendez-Gomez H., Padala S., Grubb M., Fillingim M., Monsalve A., Sayour E.J., Dobson J, & Mitchell D.A. (2019). Dendritic Cell-Activating Magnetic Nanoparticles Enable Early Prediction of Anti-Tumor Response with Magnetic Resonance Imaging. ACS nano, 13(12), 13884-13898.
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7

Quantification of Total Serum Iron

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Total iron was measured by Iron Assay Kit (MAK025, Sigma-Aldrich, St. Louis, MO). Briefly, whole blood was collected from mouse by cardiac puncture and serum was prepared by centrifugation. Serum was stored at −80C before experiments. Serum (5 – 10 fold dilution) was added directly to the transparent 96-well plate for chemical reaction. In the well, iron from the serum (5 – 10 fold dilution) was released by the addition of acidic buffer and reduced Fe3+ to Fe2+. Released iron was then reacted with a chromagen that can produce a colorimetric (at 593 nm) product, proportional to the total iron level. Standard curves were prepared using iron solutions provided by the kit. The blank background for the assay is the colorimetric reading for the iron solution and blank was subtracted from all readings. Total iron concentrations were determined from the standard curve. For measuring liver iron contents, mouse livers were homogenized using Liberase (Sigma-Aldrich, St. Louis, MO) followed by treatment with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA). Cell extracts were added into transparent 96-well plates for iron assay as described above. All colorimetric readings were taken by Cytation 5 multi-mode plate reader (BioTek, Winooski, VT).
Ma S., Dubin A.E., Zhang Y., Mousavi S.A., Wang Y., Coombs A., Loud M., Andolfo I, & Patapoutian A. (2021). A role of PIEZO1 in iron metabolism in mice and humans. Cell, 184(4), 969-982.e13.
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8

Quantification of Iron Levels in M. tuberculosis

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M. tuberculosis H37Ra cultures treated with DQYD were harvested by centrifugation. The cell pellets were washed twice with ice-cold PBS, resuspended with glass beads, and lysed using Mini-Beadbeater. The total iron, ferrous iron, and ferric concentration were detected according to the instruction of Iron Assay Kit (Sigma). Briefly, culture samples were rapidly homogenized in 4~10 volumes of cold iron assay buffer and centrifuged at 12,000g for 10 minutes at 4°C to remove insoluble material. Then 5 μL of iron assay buffer or iron reducer was added to the sample and mixed well. Then the reaction is incubated for 30 minutes at room temperature. Add 100 μL of iron probe to each well and incubate the reaction for 60 minutes. Finally, iron concentration was measured at the absorbance at 593 nm and calculated according to the instruction.
Tang B., Wei M., Niu Q., Huang Y., Ru S., Liu X., Shen L, & Fang Q. (2017). Antimicrobial Activity of Quinazolin Derivatives of 1,2-Di(quinazolin-4-yl)diselane against Mycobacteria. BioMed Research International, 2017, 5791781.
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9

Lipid ROS and Iron Quantification

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C11‐BODIPY (Thermo Fisher) was used to detect lipid reactive oxygen species (ROS). Total intracellular iron and ferrous iron (Fe2+) were measured using an iron assay kit (Sigma–Aldrich). Further details are provided in Supporting Information File S1.
Yang J., Sun L., Liu X., Huang C., Peng J., Zeng X., Zheng H., Cen W., Xu Y., Zhu W., Wu X., Ling D., Zhang L., Wei M., Liu Y., Wang D., Wang F., Li Y., Li Q, & Du Z. (2023). Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells. Clinical and Translational Medicine, 13(9), e1423.
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10

Iron Quantification in Tumor Samples

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Ferrous concentration was determined from tumor specimens of indicated groups using the manufacturer’s protocols of the Iron Assay Kit (Sigma-Aldrich, MAK025), which was previously reported in (26 (link)).
Mukhopadhyay S., Encarnación-Rosado J., Lin E.Y., Sohn A.S., Zhang H., Mancias J.D, & Kimmelman A.C. (2023). Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer. Science Advances, 9(16), eadf9284.
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