*2.8. Cell Culture*

Rat C6 glioma cell and human cervical cancer cell lines (HeLa, SiHa, and CaSki) were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were routinely maintained in Dulbecco's modified Eagle´s medium (DMEM) supplemented with fetal bovine serum (Gibco BRL) with 5% for C6 cells and 10% for cervical cancer cells, and incubated at 37 ◦C in an atmosphere comprising 5% CO2 and 95% air at high humidity. Cells were harvested with 0.025% trypsin and 0.01% EDTA (Gibco BRL).

The effect of EEP, cisplatin, and temozolomide on the proliferation of cells were evaluated using the MTT assay (3-(3,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium), which is based on the reduction of a tetrazolium salt in metabolically active cells. The procedure was as follows. Viable cells were seed into 96-well plates in 100 μL per well of DMEM culture medium at a density of 3 × 10<sup>3</sup> for C6 cells, 2 × 10<sup>4</sup> for CaSki cells, and 1 × 10<sup>4</sup> cells for HeLa and SiHa cells. After treatment, the medium was removed and the MTT solution was added to each well, followed by 1 to 2 h in a humidified atmosphere containing 5% CO2 at 37 ◦C. The absorbance of the samples was measured spectrophotometrically at λ 570 nm using a microtiter plate ELISA reader. Results are expressed as the percentage of MMT reduction.

C6 cells were exposed for 72 h with 2 to 200 μg/mL of EEP, since it is the time used to make the exposure with temozolomide (first line treatment used for glioblastoma), whereas HeLa, SiHa, and CaSki cells were exposed for 24 h with 15 to 500 μg/mL of EEP; after that time, cell viability was quantified using the MTT assay. Temozolomide (250 μM) and cisplatin (5–320 μM) were used as a control. The concentration of drugs to reach 50% growth inhibition (IC50) was obtained from the survival curves. The experiments were conducted in triplicate in independent experiments. Values are expressed as the mean ± SEM of at least three independent experiments. SigmaPlot 12.3 software (Systat Software, Santa Clara, CA, USA) was used.
