Iron assay kit

Lipid peroxidation and iron accumulation are the two main causes of ferroptosis [16 (link)]. Therefore, we measured malondialdehyde (MDA), a lipid peroxidation end product [17 (link)], and glutathione peroxidase 4 (GPX4), a phospholipid hydroperoxidase that inhibits lipid peroxidation [18 (link)]. Glutathione (GSH) is an antioxidant, and its depletion induces lipid peroxidation by activating lipoxygenases and inhibiting GPX4 activity [19 (link)]. To eliminate cellular debris, the lung tissues were homogenized, sonicated, and centrifuged at 500× g for 20 min at 4 °C. As biomarkers of ferroptosis, the Fe²⁺ level, MDA level, GSH level, and GPX4 activity were measured in lung tissues using an iron assay kit (catalog no. E-BC-K139-M, Elabscience, Houston, TX, USA), MDA assay kit (catalog no. MBS2605193, MyBioSource, San Diego, CA, USA), GSH assay kit (catalog no. MBS267424, MyBioSource), and GPX4 enzyme-linked immunosorbent assay kit (catalog no. MBS934198, MyBioSource), respectively, according to the directions provided by the respective manufacturers.

The assay was performed following published methods32 (link). Malondialdehyde (MDA) was quantified using a lipid peroxidation assay kit (Abcam, UK). After adding TBA to standards and samples, the mixtures were incubated at 95°C for 1 h, followed by a 10 min ice bath. Absorbance was measured at 532 nm using a microplate reader.
Iron was quantified with an Iron Assay kit (ElabScience). Homogenized samples in iron assay buffer were centrifugated at 16,000 ×g and 4°C for 10 min. The supernatant (10 µL) was mixed with 90 µL iron assay buffer and then incubated with 5 µL iron reducer at 25°C for 30 min. Finally, each mixture was incubated with an iron probe in the dark, and absorbance was measured at 532 nm.
Glutathione (GSH) was measured using a commercial glutathione assay kit (Elabscience, CHN). A 5% 5-sulfosalicylic acid solution was used to prevent GSH autoxidation and degradation. After lysing, 10 µL of the supernatant was incubated with the reaction mix. Finally, GSH content was determined by measuring absorbance at 405 nm.

Iron Assay Kit (Elabscience) was used to determine the concentration of Fe²⁺ in KLE cells after indicated treatment. Briefly, KLE cells were homogenized with iron assay buffer on ice and then centrifuged at 16,000 × g for 15 min at 4°C to collect lysis supernatant. Finally, a 100 μl lysis sample was incubated with 5 μl iron reducer for 30 min at 37 °C, followed by the addition of 100 μl iron probe. The mixture was incubated in the dark for 30 min at 37 °C. The absorbance was measured at 593 nm by a microplate reader (Bio-Tek Instruments, Inc.).