Quantification of Intracellular Ferrous Iron

Ferrous iron (Fe²⁺) was measured using an iron assay kit (Sigma) and a FerroOrange probe (Dojindo Laboratory). For the iron assay kit, iron is released by the addition of an acidic buffer and then reacted with a chromogen, resulting in a colorimetric product (593 nm), which is directly proportional to the Fe²⁺ concentration. The absorbance at 593 nm was measured using a plate reader (Bio-Rad). The FerroOrange probe was used for fluorescence imaging of intracellular Fe²⁺ (54 (link)). Briefly, FerroOrange (1 mM) dispersed in serum-free medium was added to the cells, followed by incubation for 30 min at 37°C. The cells were then fixed with 4% paraformaldehyde for 45 min and permeabilized with 0.2% Triton X-100 for 20 min. After that, the cells were blocked with phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) for 1 h and then incubated with anti-HSV-1 gD antibody (1:1,000 in 5% BSA) overnight, followed by staining with FITC-labeled goat anti-mouse IgG (ABclonal) (1:1,000 in 5% BSA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) for 5 min at 37°C in the dark. Cells were photographed under a confocal microscope (A1R; Nikon, Japan). The fluorescence intensity was analyzed using ImageJ software.