Measurements of mitochondrial iron in vivo.

Mitochondria lysates were prepared from mitochondria derived from heart samples by centrifugation. Briefly, LV tissues were homogenized in HES buffer (10 mM Hepes-NaOH, 0.25M Sucrose, and 1 mM EDTA-2Na) and then centrifuged at 4000 g for 10 minutes at 4°C. Supernatants were collected, and mitochondria were isolated as pellets by centrifuging them at 13,000 g for 10 minutes at 4°C. Isolated mitochondria were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (25 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Mitochondrial iron quantification was conducted using Metalloassay Kit Ferrozine method (Metallogenics, FE31M). Mitochondrial lysates were mixed with 6M HCl until the pH reached 2 and were then spun down to obtain the supernatant. Samples were mixed with R-A buffer, and the baseline absorbance at 560 nm of the samples was determined (OD1) using Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Then, R-R Chelate Color was added to the samples, and after 5 minutes of reaction, the absorbance at 560 nm was again determined (OD2). To measure iron concentration in the samples, the increase in absorbance, which is OD2−OD1, was calculated and compared with the standard curve.