Iron deposits of DCs and FTH-DCs were examined by Prussian blue staining and cellular iron measurement. In brief, cells were incubated in medium supplemented with ferric ammonium citrate (FAC, 25 μM and 250 μM) for 10 h. After washing with PBS twice cells were fixed with 2% PFA at 4°C for 1 hour and incubated with staining solution by mixing equal volumes of 5% potassium ferrocyanide and 5% hydrochloric acid for 30 min. Counterstaining was performed with nuclear fast red for 5 min.
Cellular iron levels were measured using the Total Iron Reagent Set (Pointe Scientific, Canton, MI, USA). Briefly, cells were incubated in 25 μM and 250 μM FAC-supplemented medium for 10–24 h, rinsed with PBS, and enumerated. Cells were collected, resuspended in 6N HCl solution, and incubated at 70°C for 30 min. According to the manufacturer's recommendations, total iron levels were measured, and average iron concentrations in cells were calculated by dividing total mean values by cell number.
Cellular iron levels were measured using the Total Iron Reagent Set (Pointe Scientific, Canton, MI, USA). Briefly, cells were incubated in 25 μM and 250 μM FAC-supplemented medium for 10–24 h, rinsed with PBS, and enumerated. Cells were collected, resuspended in 6N HCl solution, and incubated at 70°C for 30 min. According to the manufacturer's recommendations, total iron levels were measured, and average iron concentrations in cells were calculated by dividing total mean values by cell number.