Antibiotic mixture

U-373 MG (Uppsala; p53 mutant) and U-87 MG (p53 wild type), human glioblastoma astrocytoma cell lines derived from a malignant tumor by explant technique, were provided by ECACC cell collection (Salisbury, UK). The cells were cultured in EMEM (Eagle’s Minimal Essential Medium) supplemented with 2 mM glutamine, 1% non-essential amino acids (NEAA), 1 mM sodium pyruvate, 10% FBS (fetal bovine serum), 1% antibiotic mixture (Sigma-Aldrich, St. Louis, MO, USA), in a 5% CO₂ humidified incubator at 37 °C. Human glioma stem-like cells (GSCs) were purchased by AcceGen Biotechnologies (USA) and cultured in the specific human glioma cancer cell medium provided by the same company. The media were renewed every 2 days and confluent cells were trypsinized, subdivided, and re-plated. In these experiments, U373 and U87 cell lines were used from passage 5 to 10, and the GSCs were used from passage 3 to 6. In addition, the expression of two genes associated with GSCs, CD133 and OCT4 (octamer-binding transcription factor 4), was examined at the passage 6 by RT-PCR; Western Blot analysis was also performed to evaluate protein expression. The amplified PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining.

Normal human glial cell line HEB and glioma cell lines T98G, U87, SHG44, and U251 were bought from Tongpai Biotechnology (Shanghai, China). DMEM with high glucose and sodium glutamate (Hyclone, GE, Marlborough, USA), added with 10% FBS (Gibco, Invitrogen, California, USA) and 1% antibiotic mixture (Sigma-Aldrich, St. Louis, USA), was applied to culture cells. All cells were cultivated in a 37°C incubator (Panasonic, Osaka, Japan) containing 5% CO₂.

Mesothelioma cells were isolated from pleural biopsy specimens. The tissue was finely minced with a cutter, incubated with a digestion solution composed of 0.5% trypsin (Sigma-Aldrich, Milan, Italy) and 50 µg/ml DNase I (Roche, Milan, Italy) in Hanks’ Balanced Salt solution with Ca²⁺Mg²⁺ 0.5 mM (Sigma-Aldrich) overnight at 4°C. Next, the enzymatic solution was replaced with collagenase type 1 (3 mg/ml) (Worthington Biochemical Corporation, DBA) diluted in Medium 199 with Hank’s salts (Euroclone Spa, Milan, Italy) for 30 min at 37°C. The digestion was blocked with 10% fetal bovine serum (FBS, GIBCO, Life Technology) and the cell suspension was filtered through a 100 µm pore filter (BD Biosciences, Italy).
The cells were seeded in a 12.5 cm² flask and cultured using Roswell Park Memorial Institute (RPMI) medium 1640 with GlutaMAX (Life Technologies, Milan, Italy), 45% human endothelial cells serum-free medium (HESF, Life Technologies), 10% heat-inactivated FBS supplemented with EGF (5 ng/ml), basic FGF (10 ng/ml), and penicillin–streptomycin (Sigma-Aldrich). Fresh medium was replaced every 2–3 days. MES were used at their five to eight passages for all the in vitro experiments.
Met5A cells were purchased from ATCC. These cells were grown in DMEM supplemented with 10% FBS and 1% antibiotic mixture (Sigma-Aldrich) and maintained at 37°C in humidified atmosphere with 5% CO₂.

HCT116 cell tumorspheres were formed following the modified protocol of Feng et al. (2012) [79 (link)]. Briefly, 1 mL of 1% agarose (w/v) was added to 6-well plates. Once the agarose had gelled, HCT116 cells (2 × 10³) were grown in 2 mL of DMEM-F12 supplemented with 10% BSA (Sigma-Aldrich), 2% B27 supplement (Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/mL of EGF and b-FGF, and 1% antibiotic mixture (Sigma-Aldrich). Tumorspheres were obtained after a minimum period of 10 days, with medium replacement every 5 days. Images were taken with an Olympus CKX41 inverted light microscope (Olympus Corporation, Tokyo, Japan) and tumorspheres were collected from plates for RNA extraction in order to test the expression of genes associated with the CSC phenotype by real time PCR and a viability assay.

MM.1S (steroid-based therapy-resistant) and MM.1R (dexamethasone-resistant), human B lymphoblasts obtained from the peripheral blood of a patient affected by MM, were provided by ATCC^® CRL-2974^™ and ATCC^® CRL-2975^™ (ATCC Manassas, Manassas, VA, USA), respectively. Both cell cultures were plated in RPMI-1640 media (ATCC Manassas, Manassas, VA, USA) with 2 mM L-glutamine, 1 mM sodium pyruvate, 1% antibiotic mixture (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS) (ATCC Manassas, Manassas, VA, USA) in a humidified incubator at 37 °C with a percentage of 5% CO₂. In addition, RPMI 1788 cells (ATCC^® CCL-156™; ATCC Manassas, Manassas, VA, USA), which are human B lymphoblasts obtained from the peripheral blood of a healthy donor, were cultured in RPMI-1640 medium supplemented with 20% FBS (ATCC Manassas, Manassas, VA, USA) and a 1% antibiotic mixture (Sigma-Aldrich, St. Louis, MO, USA) in a humidified incubator at 37 °C with a percentage of 5% CO₂.