Dulbecco’s Modified Eagle’s Medium (DMEM)

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	supplemented with 1% sodium pyruvate (Sigma), 1.5 g/L sodium bicarbonate, 10% fetal bovine serum and 1% antibiotics, including 1% penicillin/streptomycin and 25 ug/ml amphotericin B (Sigma) at 37 °C in 5% CO2

Protocol tips
supplemented with 1% sodium pyruvate (Sigma), 1.5 g/L sodium bicarbonate, 10% fetal bovine serum and 1% antibiotics, including 1% penicillin/streptomycin and 25 ug/ml amphotericin B (Sigma) at 37 °C in 5% CO2

Publication protocol

The Hep G2 and LCL-PI 11 cells were cultured with DMEM (pH 7.2–7.4) supplemented with 1% sodium pyruvate (Sigma), 1.5 g/L sodium bicarbonate, 10% fetal bovine serum and 1% antibiotics, including 1% penicillin/streptomycin and 25 ug/ml amphotericin B (Sigma) at 37 °C in 5% CO2 to promote attachment. When the cells reached > 80% confluence, 5 × 105 cells were seeded into 96-well plates, allowed to adhere for 24 h and subsequently treated with medium containing different doses of E2 (0.01, 0.1, 1, 5 and 10 μM/lit) to determine the viability and IC50 values. After 24, 48, 72 and 96 h of the treatment, the effect of E2 was assessed by MTT assay according to standard protocols. In this regard, the Hep G2 and LCL-PI 11 cells were washed twice with PBS, and a fresh medium containing MTT (0.5 mg/mL) was added and finally, after 4-hour incubation, the formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm. All experiments were repeated three times, with at least three measurements (triplicates).

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