4.4. Intracellular Iron Quantification

The total amount of iron taken up by the cells was determined by a colorimetric assay. The detailed protocol for conducting the modified ferene-s measurement of iron associated with cells after exposure to BNF nanoparticles has been previously described [29]. The stock reagent, working reagents, and standard reference materials were prepared beforehand. Ammonium acetate and glacial acetic acid were purchased from ThermoFisher Scientific Corporation (Waltham, MA, USA). The Fe standard reference material (SRM), FeCl₃, Iron Standard for ICP (Inductively Coupled Plasma Spectroscopy), and 1000 ± 2 mg/L Fe in 2% nitric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA), for calibrating the ferene-s assay. Briefly, 1 × 10⁶ macrophages were suspended in 1 mL media and were incubated at 37 °C during the different treatments with the occasional shaking or tapping of tubes to maximize distribution and prevent settling. After incubation, cells were centrifuged to separate them from free nanoparticles that remained in the supernatant, washed with PBS, and centrifuged a second time. This washing with PBS was repeated three additional times. The final cell pellet was resuspended in 1 mL of PBS and counted using a cellometer to estimate the total number of cells. A known number of cells in PBS was transferred into a 1 mL Eppendorf tube and centrifuged. The supernatant was discarded, and 1 mL of working solution was added to the cell pellet. Cell pellets were digested in the working solution by incubating them at room temperature for at least 20 h. Tubes were then centrifuged to separate solid cell debris, and the supernatant was analyzed with a UV/vis spectrophotometer at 595 nm. A calibration curve developed with known quantities of Fe using the SRM was used to estimate the iron concentration in the test samples.