An iron Assay kit (BioAssay Systems) was utilized to evaluate the concentration of Fe2+ in HT29 cells following treatment with oxaliplatin and the ferroptosis inducer, erastin. The experiment was performed in accordance with the recommendations of the manufacturer, and the absorbance of cells at 593 nm was measured by using a spectrophotometer (Shanghai Mapada Instruments Co., Ltd.).
Iron assay kit
Manufactured by BioAssay Systems
Sourced in United States
The Iron Assay Kit is a colorimetric assay that quantifies iron concentration in biological samples. It measures the absorbance of a chromogenic complex formed by iron and the provided reagents, allowing for the determination of iron levels.
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Lab products found in correlation
Erastin, by Apexbio (1 mentions)
P nrf2, by Proteintech (1 mentions)
Ml385, by Apexbio (1 mentions)
Antibodies against nrf2, by Proteintech (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Transwell insert, by Corning (1 mentions)
10 protocols using iron assay kit
1
Quantifying Fe2+ in HT29 Cells
2
Cellular Iron Quantification Assay
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Cellular iron level was measured with iron assay kit (BioAssay Systems). Cells were homogenised through five volumes of iron assay buffer followed by 13,000 g centrifugation for 10 min at 4°C. Afterwards, incubation of iron reducer together with supernatant mixtures was conducted for 30 min. Samples were then cultured with iron probe for 60 min away from light. The absorbance values were measured at 593 nm.
3
Ferroptosis Modulators in Oxidative Stress
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Art (purity ≥ 98%), erastin, and ML385 were purchased from APExBIO Technology LLC (Houston, USA). Streptozotocin (STZ) was purchased from Sigma‒Aldrich (USA). Antibodies against Nrf2, p-Nrf2, β-actin, heme oxygenase 1 (HO-1), and GPX4 were from Proteintech Group, Inc. (USA). The Iron Assay Kit was purchased from BioAssay Systems (Hayward, CA, USA). ROS, MDA, and GSH assay kits were from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, China).
4
Evaluating Iron and Lipid Peroxidation
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An iron assay kit (BioAssay Systems) was used to test the ferric iron level. The relative malondialdehyde (MDA) level was evaluated by a lipid peroxidation detection kit (Solarbio, Beijing, China) according to the manufacturer's instructions.
5
Quantification of Iron Levels in Biological Samples
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Non-heme and heme iron assays were performed as previously described [48 (link)]. In brief, blood samples were collected from anesthetized mice through intracardiac puncture. Tissue and cell samples were lysed in RIPA Lysis Buffer (Beyotime Biotechnology), and protein concentrations were measured using the BCA Protein Assay (Beyotime Biotechnology). Detection of non-heme iron content was measured using an iron assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer's instructions and normalized to the protein concentration of each sample. Calculation of iron release was performed as previously described [49 (link)]. To determine heme levels, equal amounts of samples were mixed with 2 M oxalic acid (Meilunbio, Beijing, China), heated to 95 °C for 30 min, and then centrifuged at 1000 g for 10 min at 4 °C to remove debris. The fluorescence of the supernatant was assessed on the Synergy 2 Multiscan Spectrum (BioTek, Vermont, USA) and normalized to the protein concentration of each sample.
6
Mitochondrial Iron Content Quantification
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Isolated mitochondria collected using the Mitochondrial Isolation Kit for Tissue (Pierce, Rockford, IL) were diluted with EDTA-free buffer after sonication. Mitochondrial non-heme iron contents were measured with a commercial Iron Assay Kit (BioAssay Systems, Hayward, CA), which directly detects total iron in the sample, according to the manufacturer’s protocol16 (link). Isolated mitochondria were sonicated and diluted with EDTA-free buffer, and then iron concentrations were normalized to the mitochondria concentration of each sample.
7
Quantifying Hepatic Iron Levels
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Blood samples collected from anesthetized mice through intracardiac puncture. Livers were harvested, weighed, mechanically homogenized in RIPA Lysis Buffer and mixed with HCl (0.01 M final concentration). Iron content was detected using an iron assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions.
8
Macrophage-HepG2 Co-culture System for Iron Study
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THP-1 cells were differentiated into adherent macrophage-like cells by 100 nM phorbol 12-myristate 13-acetate (PMA; MilliporeSigma) for 48 h. A non-contact culture system was set up following a published protocol21 (link),22 (link) In brief, THP-1 macrophages were seeded at 5 × 105 cells per well on the top surface of the Transwell inserts (Corning-Costar, Corning, NY, USA). HepG2 cells were grown in 6-well plates. On the day of infection experiments, HepG2 cells were overlaid with Transwell inserts containing THP-1 macrophages, and the bacterial suspension was added to the apical side of the Transwell system. To assess iron content of macrophages in this co-cultured system, THP-1 macrophages were incubated with 100 μM ferrous chloride (Adamas, Shanghai, China) for 16 h prior to be placed above HepG2 cells.22 (link) The relative iron concentration in co-cultured macrophages lysates was determined using an iron assay kit (BioAssay Systems).
9
Iron Quantification in Cells
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Iron concentration was measured by Iron Assay Kit (BioAssay Systems, USA). The procedure was performed as described [26 (link)] with minor modifications. The cells were crushed with soniprep (Ultrasonic cell crusher, SONICS, USA) after washing with PBS. Samples were incubated for 40 min at room temperature, and optical density was read at 590 nm. The results were expressed as fold change.
10
Measurement of Mitochondrial Iron
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After collecting isolated mitochondria using Mitochondrial Isolation Kit for Tissue (Pierce, MD, USA), mitochondrial iron contents were measured using a commercial Iron Assay Kit (BioAssay Systems, CA, USA) according to the manufacturer’s protocol [41 (link)].
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