2.4. Cell Culture, Palmitate Treatment, and Cell Viability

INS-1, known as a murine pancreatic β-cell line, was sub-cultured in RPMI 1640 medium (Paisley, Gibco, UK) containing 1% penicillin/streptomycin (Welgene, Daegu, Korea) and 10% fetal bovine serum (Gibco) in an atmosphere of 5% CO₂ at 37 °C. To facilitate the absorption of saturated fatty acids into cells, palmitic acid (PA, Sigma) was prepared by dissolving 20 mM PA in 10 mM NaOH at 70 °C for 30 min, and then conjugated with 5% BSA solution dissolved in DPBS at a ratio of 1:3. After seeding at 2.5 × 10⁴ cells/well of INS-1 cells for overnight, the cells treated with 0.4 mM palmitic acid with or without 500 µg/mL ADLE or 25, 50, and 100 µg/mL fractions for 24 h. Cell viability was assayed by MTT method (Duchefa Biochemie BV, Haarlem, The Netherlands). At the end of the reaction, after removing the supernatant, MTT at 1mg/mL was added to well by 100 μL each, and the cells were incubated for 2 h. Once completed, purple insoluble formazan crystals were dissolved by 200 μL of 2-propanol and measured at 540 nm (TECAN Group Ltd., Shanghai, China).