C6 glioma cells

Reagents, chemicals and media were obtained from commercial suppliers and used without further purification: FluoZin-3 AM and TSQ (N-(6-Methoxy-8-Quinolyl)-p-Toluenesulfonamide) were purchased from Molecular Probes, Invitrogen. Ethylenediaminetetraaceticacid disodium calcium salt (CaEDTA), N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and 2-mercaptopyridine N-oxide (pyrithione) were obtained from Sigma-Aldrich. All other chemicals and media like Dulbecco’s Modified Eagle’s Medium/Nutrient mixture F12 Ham (DMEM/F12, 1:1), FBS, Penicillin, Streptomycin and Amphotericin B were purchased from HiMedia; C6 Glioma cells from ATCC.

C6 glioma cells, a rat cell line of astrocytic origin, were purchased from the American Type Culture Collection (Rockville, MD, USA). The primary rat astrocyte cell line was a generous gift from Dr. Jiahn-Chun Wu (National Yang-Ming University, Taiwan) [28 (link)]. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (both from Gibco BRL, Grand Island, NY), 1 mM sodium pyruvate (Sigma, St. Louis, MO, USA), and 100 IU/mL penicillin and streptomycin (pH 7.2) (Gibco BRL, Grand Island, NY). Cells were incubated in a humidified atmosphere of 5% CO₂/95% air at 37°C.

Four Sprague—Dawley (SD) rats weighing 200–280 g were used to create rat models of brain tumor. All rats were housed in standard facilities and provided ad libitum access to water and rodent food. The animal experiments were performed according to guidelines of the Chinese Animal Welfare Agency and were approved by the Institutional Animal Care and Usage Committee of Shantou University Medical College. We had a protocol in place for the early euthanasia/humane endpoints for animals who became severely ill/moribund during the experiment(s). C6 glioma cells were obtained from American Type Culture Collection (ATCC, USA) and grown in Dulbecco’s modified Eagle medium (DMEM/F12, Gibco, USA) with 10% fetal calf serum and penicillin/streptomycin under a 5% CO₂ atmosphere at 37°C. Each rat was injected with 1 × 106 exponential-phase C6 glioma cells in the right basal ganglia [20 (link)]. To alleviate surgical pain, preemptive analgesia (Buprenorphine 0.05 mg/kg, S.C.) was administered 30 min before operation and post-procedural analgesia (Buprenorphine 0.05 mg/kg, SC q 6–12 hr) was continued for a minimum of 12–24 hours after the animal regained consciousness. After operation, the rats were returned to their home-cage, no cage contained two rats to minimize any enhanced intermale aggression.

In the cellular uptake experiments, F98, C6 rat glioma, and 9L rat gliosarcoma cell lines were used. Each cell was provided by or purchased from as follows: F98 glioma cells, Dr. Rolf Barth (Ohio State University, Columbus, OH, USA); C6 glioma cells, the Japan Collection of Research Bioresources (JCRB) Cell Bank, National Institute of Biomedical Innovation (Osaka, Japan); 9L rat gliosarcoma cells, the American Type Culture Collection (ATCC; Manassas, VA, USA). All the cells were cultured in the medium that was used in our laboratory [16 (link),32 (link),33 (link),34 (link),35 (link),36 (link)]. This medium contained Dulbecco’s modified Eagle’s medium (DMEM) and was supplemented with 10% fetal bovine serum, penicillin, streptomycin, and amphotericin B. All the materials for making this culture medium were purchased from Gibco Invitrogen (Grand Island, NY, USA). Of these cell lines, F98 glioma cells are particularly useful in evaluating therapeutic agents [16 (link),32 (link),33 (link),34 (link),35 (link),36 (link)] because they simulate the behavior of human malignant gliomas in intracerebral implantation, including their high-infiltrative growth pattern and low immunogenicity [37 (link),38 (link)]. The F98 glioma-bearing rat brain tumor model was used for in vivo evaluation.