Dulbecco s modified eagle s medium nutrient mixture f 12

ARPE-19 cells (Cobioer Biosciences, Nanjing, China) were cultured in Dulbecco's modified Eagle's medium/Nutrient mixture F12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5 mM HEPES buffer, 7.5% NaHCO₃, 10% fetal bovine serum, and 1% penicillin/streptomycin; cells were maintained at 37°C and 5% CO₂.
To investigate the impacts of exosomes from different patient groups on VEGF expression, ARPE-19 cells were treated with 50 μg of exosomes. During repetitive exosome treatment, ARPE-19 cells were treated three times at 24-hour intervals. After treatments, ARPE-19 cells were harvested for real-time polymerase chain reaction and western blotting analysis.
To investigate the impacts of miRNAs on RPE proliferation, ARPE-19 cells (transfected as described in the next section) were seeded in 96-well plates (1 × 10⁴ cells/well) and incubated for 0, 1, 2, 3, 4, and 5 days at 37°C. Subsequently, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/mL) was added at the indicated growth intervals and the cells were incubated for an additional 4-hour interval. Dimethyl sulfoxide (100 µL) was then added to each well. Finally, the absorbance was measured at 490 nm using a standard microplate reader.

The adult neurosphere lines GSC-11 and GSC-23, a kind gift of Dr. Lang at UT MD Anderson Cancer Center, were established from acute cell dissociation of human GBM surgical specimens and maintained in our lab. The neurosphere lines GBM1A and GBM1B were originally derived and characterized by Vescovi and co-workers [12 (link)]. All neurosphere cell lines were cultured and maintained in serum-free medium containing Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (1:1, vol/vol) (Thermo Fisher Scientific Inc, Waltham, MA) supplemented with 1% of Penicillin/Streptomycin (Lonza, Verviers, Belgium), B27 10x (Thermo Fisher Scientific Inc, Waltham, MA) and 20 ng/ml of both EGF and FGF (Sigma-Aldrich, St Louis, MO) according to the procedures described by Galli [12 (link)].
The human embryonic kidney 293FT (HEK293FT) cell line was purchased from ATCC and were maintained in Dulbecco’s modified Eagle/F12 medium (1:1, vol/vol) and supplemented with 10% FBS (Fetal Bovine Serum, Thermo Fisher Scientific Inc, Waltham, MA). All the cell lines were grown at 37°C in a humidified incubator with 5% CO2. All cell models were authenticated by STR sequencing.

BMSCs were isolated and cultured as described by Huang et al (15 (link)) with some modifications. In addition, all attempts were made to minimize rat suffering. Briefly, following sacrifice, the femurs and tibias of rats were quickly stripped, and muscle and extraossial tissue were trimmed. A 5 ml syringe equipped with complete culture medium [Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 U/ml streptomycin] was inserted into the bone marrow cavity, so as to flush target bone marrow cells into culture dishes, which were cultured in an atmosphere containing 5% CO₂ at 37°C. The medium was initially substituted at 48 h and was then changed every 3 days. Once the cells reached 80% confluence, adherent cells were trypsinized with 0.25% trypsin solution and passaged. Cells from passages 3-5 were available for use in the following experiments.

Human dermal fibroblasts (HDF) were obtained from the diagnostic skin punch biopsies following a published protocol [68 (link)]. For in vitro experiments, half of the skin biopsy from the lower leg was used. After mechanical separation of dermis and epidermis, dermal tissue was cultivated in fibroblast expansion medium (Dulbecco's modified eagle's medium/Nutrient Mixture F-12 (Thermo Fisher Scientific, Waltham, MA, USA), 10% fetal calf serum (FCS, Biochrom, Berlin, Germany), 100 U/ml penicillin/streptomycin (Pen/Strep, Thermo Fisher Scientific, Waltham, MA, USA)) under 5% CO₂ and 37 °C [34 (link)].