2.5. Tissue Iron Measurements

Iron levels in brain and spleen samples were measured by Agilent 8900 triple quadrupole Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Santa Clara, CA, USA). Briefly, 5 µL of 67% nitric acid (Thermo Fisher Scientific, Waltham, MA, USA) per mg brain sample was used to lyse the tissue overnight at room temperature, followed by a 1 h digestion at 90 °C using 5 µL of 30% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) per mg brain tissue. For spleen samples, 10 µL of 67% nitric acid(Sigma-Aldrich, St. Louis, MO, USA) per mg spleen sample was used for lysis overnight at room temperature, followed by a 1 h digestion at 90 °C using 10 µL of 30% hydrogen peroxide per mg spleen tissue. Both brain and spleen samples were further diluted (1:5) with 1% nitric acid before iron detection by ICP-MS. Calibration curves were drawn from calibration blanks at six standard points with different iron concentrations (4 nM–4 µM). For the acute dosing study, brain iron levels were determined in the saline (n = 3) and TfRMAb (n = 3) treated mice. Spleen iron levels were not determined following acute dosing. For the chronic dosing study, brain and spleen iron levels were determined in saline (n = 4) and TfRMAb (n = 4) treated mice. Brain and spleen iron levels were not determined in the mouse IgG1 treated mice.