Dulbecco s mem

Mouse neuroblastoma Neuro2A cells (ATCC-CCL 131) were grown in Dulbecco’s MEM (Life Technologies) containing 10% fetal bovine serum with 100 units/mL penicillin and 100 mg/mL streptomycin (Life Technologies). The cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂ and 95% air. For measurement of ROS generation, the cells were seeded at 2.0 × 10⁴ cells/well in poly-D-lysine-coated 96-well plates (BD Falcon, Franklin Lakes, NJ, USA). After plating, the cells were maintained in Dulbecco’s MEM (without phenol red or serum, Life Technologies) with 100 units/mL penicillin and 100 mg/mL streptomycin for more than 16 hours until subsequent experiments. Intracellular ROS generation caused by oxaliplatin in neuro2A cells was determined using CM-H₂DCFDA. CM-H₂DCFDA is converted by an intracellular esterase into 2’,7’-dichlorodihydrofluorescein (DCFH). The non-fluorescent DCFH is then oxidized by ROS to the highly fluorescent compound 2’,7’-dichlorofluorescein. The cells were incubated for 30 minutes with 10 μM CM-H₂DCFDA at 37 °C, followed by treatment of drugs for 1 hour. Fluorescence intensity was measured using a microplate reader (Infinite M200; Tecan Japan Co., Ltd., Kanagawa, Japan) at 485 nm (excitation) /520 nm (emission).

HRT-18G (ATCC CRL-11663) cells, derivative of HRT-18 (ATCC CCL-244, ileocecal colorectal adenocarcinoma) were maintained in Dulbecco’s MEM (Life Technologies, Warsaw, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Life Technologies, Warsaw, Poland), penicillin (100 U/mL), streptomycin (100 μg/mL), and ciprofloxacin (5 μg/mL). Cells were cultured at 37 °C under 5% CO₂.
To generate human airway epithelium (HAE) cultures, primary cells isolated from patients’ tissue were cultured on plastic in Bronchial Epithelial Growth Media (BEGM) medium for 1–2 weeks. Next, passage 2 cells were seeded on permeable Transwell insert supports (12 mm) and after the confluence was reached, apical medium was removed, and cells were grown for another 5–8 weeks on air–liquid interface (ALI) in ALI medium. After this time, cells were fully differentiated, forming pseudostratified mucociliary epithelium.

The swine kidney cell line SK-L [15 (link)] was propagated in Eagle’s minimum essential medium (MEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 0.295% tryptose phosphate broth (Becton Dickinson, San Jose, CA, USA), 10 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (Sigma-Aldrich, St. Louis, MO, USA) and 10% horse serum (Life Technologies, Carlsbad, CA, USA). The SK6-MxLuc cell line carrying a Mx/Luc reporter gene [16 (link)] was propagated in MEM supplemented with 0.295% tryptose phosphate broth and 7% horse serum. The human embryonic kidney cell line 293T was maintained in Dulbecco’s MEM (Life Technologies) and 10% fetal calf serum (Cambrex, Grand Island, NY, USA). All cells were incubated at 37 °C in the presence of 5% CO₂.