Skanlt for multiscan fc software

The Cell Ferrous Iron Colorimetric Assay Kit (E-BC-K881-M, Elabscience, Wuhan, China) was used to determine the intracellular ferrous iron level. Approximately 1 × 10⁶ cells were extracted and homogenized on ice for 10 min with 200 μL of lysis buffer before being centrifuged at 15,000× g for 10 min to collect the supernatant. Then, 80 μL of supernatant was treated with the iron probe or the control reagent for 10 min at 37 °C. A Multiscan FC plate reader was used to measure the absorbance at 593 nm, which was then analyzed using Skanlt for Multiscan FC software (Thermo Scientific, Waltham, MA, USA). The relative ferrous iron level equals the difference between the experimental and control groups’ ferrous iron contents. A cell ferrous iron standard curve was used to calculate the cell ferrous iron content.

The cell viability was determined using CCK-8 assay kits (Dojindo Laboratories, Kumamoto, Japan). Cells were plated in a 96-well cell culture plate at a density of 5 × 10³ cells per well and cultured in growth media for 24 h before being transiently transfected with plasmids. After another 48 h at 37 °C, the cells were rinsed with PBS solution. The medium was changed for a new serum-supplemented medium. The cells were then treated for 40 min with the CCK test solution at the specified dose. A Multiskan FC plate reader was used to measure the absorbance at 450 nm, which was then analyzed using Skanlt for Multiscan FC software (Thermo Scientific, Waltham, MA, USA).

The level of thiobarbituric acid reactive substance (TBARS) was evaluated using the Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime Biotechnology, Shanghai, China). Cells were planted in 6 mm plates at a density of 2 × 10⁶ cells per plate. After 24 h of drug treatment, cells were washed with ice-cold 1 × PBS, lysed with RIPA on ice, and centrifuged at 15,000× g for 10 min to remove insoluble material. The BCA protein assay kit (23250, Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein content. The supernatant (100 μL) was incubated for 15 min at 100 °C with 200 μL of MDA test detection solution before being cooled to room temperature. The supernatant was collected by centrifuging the mixture at 1000× g for 10 min. Then, 200 μL of supernatant was transferred to a 96-well plate, and absorbance at 532 nm was recorded using a Multiskan FC plate reader and analyzed using Skanlt for Multiscan FC software (Thermo Scientific, Waltham, MA, USA). The TBARS concentration was estimated in nmoL/mg protein.