Iron tibc reagent kit

Serum iron was measured using the Iron/TIBC Reagent kit (I7506‐60; Pointe Scientific). Tissue non‐heme iron was measured using the standard chromogen method as described previously.^[41] In brief, homogenized animal tissues were incubated in NHI acid solution (10% trichloroacetic acid in 3 m HCl) at 65–70 °C for 72 h. After centrifugation to pellet the debris, 10 µL supernatant was loaded into each well in a 96‐well plate; 200 µL working solution containing 0.2% thioglycolic acid and 0.02% disodium‐4,7‐diphenyl‐1,10‐phenanthroline disulfonate in 50% saturated NaAc solution was added to each well, mixed, and the plate was incubated at room temperature for 10 min, after which the plate was read at 535 nm using a spectrophotometer plate reader (Thermo Fisher). Iron concentration was normalized to the wet tissue weight and is presented as µg g⁻¹ wet tissue.

Hematological parameters were assessed immediately after euthanasia using a Coulter Ac·T diff Hematology Analyzer (Beckman Coulter, Gladesville, Australia). Serum iron parameters were determined using the Iron/TIBC Reagent Kit (Pointe Scientific, Canton, MI) as previously described (Frazer et al. 2017 (link)). Tissue iron levels were assayed by colorimetric assay as previously described (Torrance and Bothwell 1968 (link)).