Dulbecco s phosphate buffered saline pbs

The levels of intracellular reactive oxygen species (ROS) were measured using the chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) (Invitrogen, Eugene, OR, USA) according to the manufacturer’s guidelines.
BEAS-2B cells were plated on black clear flat bottom 96-well plates (Corning, NY, USA) at a density of 20,000 cells/well (200 µl/well; culture area 0.32 cm²/well) and grown to semiconfluency for two days. After being washed with Dulbecco's phosphate-buffered saline (PBS; Biowest, Nuaillé, France), the cells were loaded with 2.5 µM CM-DCFDA in PBS for 30 min at 37 °C. Thereafter, the loading buffer was removed, and the cells were washed with PBS. Then the cells were treated with the cellulosic material dispersions at 9 doses: 4, 8, 16, 31, 63, 125, 250, 500 and 1000 µg/ml (equivalent to 2.4, 4.9, 9.8, 19.5, 39.1, 78.1, 156.2, 312.5 and 625.0 µg/cm²). 2 mM H₂O₂ (Sigma-Aldrich Chemie, Steinheim, Germany) was used as a positive control, while untreated cells served as a negative control at each time point. Fluorescence was recorded at 3, 6 and 24 h (excitation 485 nm, emission 538 nm) using a plate reader (Fluoroskan Ascent FL, Vantaa, Finland). The average fluorescent intensity was calculated by subtracting background values. All treatments were performed in quadruplicate, and the experiments were repeated twice.

HepG2 cells (HB-8065, ATCC) were cultured in 75 cm² cell culture flasks (SPL Life Sciences, Gyeonggi, Korea); cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/low glucose (Gibco, Green Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (P/S; Gibco) at 37 °C and 5% CO₂. For subculture, HepG2 cells were washed first with Dulbecco’s phosphate buffered saline (PBS; Biowest, Nuaillé, France), and cell detachment was performed using 0.5% trypsin–EDTA (Invitrogen, Waltham, MA, USA), followed by resuspension in cell culture medium.
For the assays, 1 × 10⁶ HepG2 cells were seeded into 6-well dishes (Nunclon Delta Surface; Thermo Scientific, Waltham, MA, USA). HepG2 cells were then counted using a Neubauer-improved (0.0025 mm², depth 0.1 mm; Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) hemocytometer. After overnight incubation at 37 °C and 5% CO₂, cells were washed using PBS and resuspended in calcium-free DMEM supplemented with 2% FBS and 1% P/S, followed by incubation with 1 × 10⁷ isolated platelets for 6 h. Supernatants from each well were then collected in a 2 mL microtube, and the cells were detached using cell scrapers. All of the cells and supernatants were stored at − 20 °C until analysis.