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Then, tissue was centrifuged and digested with Trypsin EDTA solution C to generate a single cell suspension, filtered through a 40 m cell strainer, and plated in DMEM/F12 with 2.5 mM L-glutamine, 12.5 mM D-glucose, 10% FBS, penicillin (50 U/ml), and streptomycin (50 g/ml), obtaining twelve independent cell lines (three for each treatment condition)

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To ensure sustained exposure, the drinking water was refreshed twice a week, with the BAPN concentration incremented by 0.025% per change, over a duration of four weeks