Pen strep solution

Human HCC cell lines (Huh7 and PLC/PRF/5) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). All cell lines were cultivated in high glucose-DMEM containing 10% FBS (Gibco, USA) and 1% Pen-strep solution (Invitrogen, UK). Human normal liver cell lines (LO-2) were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China) and cultured in high glucose-DMEM containing 10% FBS (Gibco, USA) and 1% Pen-strep solution (Invitrogen, UK). HUVEC cells were purchased from Shanghai Institute of Cell Biology (Shanghai, China) and cultured in endothelial cell medium. All cells were cultured in a humidified 37℃ incubator filled with 5% CO₂ and then passaged when the confluence reached 90%.

Different brain structures (HT, HIP, OB, RET, CX, and CB) were collected from mice (n = 7–10 mice) of 5–7 days of age immediately after sacrifice. Tissues were collected in D-PBS and were cut into small pieces of 1 mm³. Afterwards, tissues were digested using papain digestion solution containing 20 U/mL papain (ref LS003126, Worthington Biochemical Corporation, Lakewood, NJ, USA) and 200 U/mL DNase (ref D4527, Sigma Aldrich), for 1 hour at 37°C with constant rotation. This was followed by mechanical dissociation of the pellet for 3 times using a 0.02% (w/v) BSA solution (ref A4161, Sigma Aldrich) containing 333 U/mL DNase (ref D4527, Sigma Aldrich). This was followed by cell counting using a hemocytometer and centrifugation at 1000 rpm for 10 minutes. Cells were resuspended in a glia culture medium at 1000 cells/μL and plated at 200,000 cells/glass slide. Culture medium was DMEM medium (ref 41965–039, Gibco Life technologies,) supplemented with 10% fetal bovine serum (ref 16000–044, FBS-Invitrogen/Gibco), 2 mM glutamine (Sigma, G7513), 1 mM sodium pyruvate (ref 11360–039, Invitrogen/Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Pen/Strep solution, ref 151 40–122, Invitrogen/Gibco). Cells were incubated at 37°C with 5% CO₂ for 8 days and medium was changed every 3 days.

The T24 bladder carcinoma cell line was cultured in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich). The DMEM medium was supplemented with 1% pen/strep solution (Invitrogen) and 5% fetal calf serum (FCS, Sigma-Aldrich, St. Louis, MO, USA). T24 cells were seeded in a 96-well plate (5000 cells per well) already containing DMSO as a control (0.2% final concentration) or the test compounds dissolved in DMSO. The cells were grown for five days at 37 °C in a humidified incubator containing 5% CO₂, without further change of medium, before the detection was carried out as described in [30 (link)].