Ethylene diamine tetraacetic acid (edta)

To assess drug–DNA interactions, the pPUC19 DNA plasmid (Fermentas, UK) was incubated with test compounds for different periods of time (i.e., from 15 min to 3 h). Electrophoresis was performed in 1% agarose gel for 2 h at 80 V and 55 mA in 1×  TBE buffer (0.45 M Tris borate, 0.01 M EDTA, pH 8.3, purchased from Eppendorf AG, Germany). PEQLAB electrophoresis chambers were used. Staining was performed in ethidium bromide (EtBr) solution in 1×  TBE (0.2 μg/mL) for 20 min. Images were taken under UVE light in the GelDoc-It Imaging System (UVP).

For quantification of metabolites and oxidative stress markers, eyes were enucleated and placed in prewarmed 37°C PBS. Eyes were then dissected and the whole lens carefully removed and placed in a prechilled Eppendorf tube containing 50 mM EDTA (Sigma-Aldrich, Darmstadt, Germany). For lens fractions, the epithelium, cortex, and nucleus were dissected on a prechilled petri dish and transferred to a prechilled Eppendorf tube containing 50 mM EDTA. Aqueous humour was collected as previously described.²² (link) Whole lens and fractions were then homogenized, spun down at 16,000 rcf for 20 minutes at 4°C, supernatant collected, snap frozen, and stored at −80°C. WT and xCT KO mice samples (lens and aqueous humour) for each age group were collected together and stored at -80°C for no more than 1 week. Samples for each age group were then divided into two or more batches and analyzed on different days to ensure there was no processing or instrumentation errors. WT and xCT KO mice samples were always processed together from each group.