Iron colorimetric assay kit

We measured cellular iron concentration using the Elabscience iron colorimetric assay kit. Briefly, cells were scraped off and homogenized on ice in PBS. After centrifugation, we reserved the supernatant and quantified it using a bicinchoninic acid (BCA) protein assay kit (#P0012; Beyotime). Next, we mixed 600 μL chromogenic agent with 200 μL sample and determined the OD at a wavelength of 520 nm using a Tecan Infinite 200 PRO microplate reader.

To determine whether the c2515 and c2516 are directly involved in iron uptake, we detected the iron concentrations in the WT, mutant, and complemented strains using an iron colorimetric assay kit (Elabscience Biotech, Wuhan, China). The overnight cultured strains were diluted 1:100 into 100 mL LB media containing 200 μM 2,2′-dipyridyl. Then, the strains were grown to the log phase (OD₆₀₀ = 0.6) and washed three times with PBS. Cell pellets were subsequently obtained and suspended in PBS, and the cells were lysed by ultrasonic crash to release intracellular iron. The samples were centrifugated to exclude the impurities and the supernatant was used for iron concentration determination. Deionized water (0.1 mL), iron standard stock solution (0.1 mL) was diluted according to instructions, and sample (0.1 mL) were individually mixed with a chromogenic agent (0.4 mL), boiled for 5 min, and then centrifuged at 3,000 g for 10 min. The supernatant was subsequently collected. Iron content was estimated by measuring the OD at 520 nm (OD₅₂₀) of the supernatant and a standard product curve (y = ax + b) was created. The following formula was used:

The content of GSH was detected by the corresponding commercial kit (A006-2-1, Nanjing Jiancheng Biotechnology, China) and MDA were measured using the Lipid Peroxidation MDA Assay Kit (S0131, Beyotime, China). The content of iron was measured by Iron Colorimetric Assay Kit (E-BC-K139-M, Elabscience, China). All kits were used under the manufacturer’s instructions.