2.8. Measurement of Iron Uptake into Caco-2 Cells

Iron uptake into Caco-2 monolayers was determined using cell ferritin formation (ng cell ferritin/mg cell protein). In each cell culture experiment, ferric ammonium citrate (FAC) was included as a control. FAC is a well-absorbed form of iron in Caco-2 cells and used as the reference for DMT1 uptake [18,19,20]. Reference blanks (cells not treated with iron) were included in each experiment to ensure low baseline levels of cell ferritin. After iron treatment, cells were washed twice with PBS and lysed with 200 μL CelLytic M protein lysis buffer (Sigma). Lysed cells were centrifuged (14,000× g, 15 min), supernatants collected, and analyzed for cell ferritin using the Spectroferritin ELISA kit (ATI Atlas, Chichester, UK). Cell ferritin was normalized to total protein concentrations using the BCA protein assay kit (ThermoFisher Scientific, Loughborough, UK).