Dmem f12 culture medium

5 × 10⁴ cells/well were seeded in 200 μL serum-free DMEM/F-12 culture medium in the upper chamber (8 mm pore size; Corning Incorporated, Corning, NY, USA) in the following groups: (A) control; (B) H₂O₂; (C) tBHQ; (D) tBHQ+H₂O₂; (E) tBHQ+siTrx1+H₂O₂. 500 μL complete medium was added to the lower chamber. After 24 hours, the lower chamber membrane was cleaned with PBS, placed on a 24-well plate, fixed with 95% ethanol, and stained with 1% crystal violet. After drying, the 6-well plate was imaged using a microscope (Leica Solms, Germany).

The cytocompatibility of cyclodextrin formulations was evaluated on BALB/3T3 clone A31 (ATCC^® CCL-163TM) murine fibroblasts using the WST-1 test. BALB 3T3 cells were cultured in DMEM/F12 culture medium (Corning) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Gibco). They were seeded in a 96-well plate at 1.5 × 10⁴ cells/well. To allow complete cell attachment, cells were incubated 4 h at 37 °C and 5% CO₂. Aliquots of A1 to A9 formulations, Nevanac 3 mg/mL suspension, and control (DMEM/F12) were diluted 1:50, 1:100, and 1:150 times, respectively, with complete cell culture medium to be below the IC₅₀ of nepafenac, to ensure that nepafenac was not in cytotoxic concentrations, and added to cell monolayers [45 (link)]. DMEM/F12 medium was used as control. After 24 hours of incubation with the formulations, WST-1 reagent (Roche) was added and the assay was carried out according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a Model 680 microplate reader from Bio-Rad (Hercules, CA, USA) and Microplate Manager software (Version 5.2.1, BioRad, CA, USA).