Rpmi 1640

Zinc nitrate hexahydrate (>98.0%), indole-3-Carbinol (Sigma Aldrich, St. Louis, MO, USA), sodium hydroxide (>98.0%). All media was supplied from Difco, MB Cell (Gangnam-gu, Seoul, Korea). Absolute alcohol was supplied by Samchun Pure Chemical Co. Ltd. (Gyeonggi-do, Korea). Olive oil and Tween 80 were obtained from Samchun Pure Chemical Co. Ltd. The human keratinocyte cell line (HaCaT) and lung cancer cell line (A549) used in this study were obtained from the Korean cell line bank (Seoul, South Korea). RPMI 1640 (Roswell Park Memorial Institute Medium) culture medium was purchased from GenDEPOT Inc. (Barker, TX, USA). Dulbecco’s modified eagle’s medium (DMEM) (Gibson-BRL, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (p/s) (WElGENE Inc., Daegu, Korea) was also used for experiments.

Zinc nitrate hexahydrate (>98%) and NaOH (>98%) were supplied by Dae-Jung Chemicals and Metals Co., Ltd. (Pyeontaek, Korea). Absolute alcohol, Tween 80, and olive oil were purchased from Samchun Pure Chemical Co. Ltd. (Gyeonggi-do, Korea). The human keratinocyte cell line (HaCaT) and lung cancer cell line (A549) used in this study were attained from the Korean cell line bank (Seoul, Korea). Cell culture reagents such as RPMI 1640, DMEM, penicillin–streptomycin, and fetal bovine serum (FBS) were obtained from Gen DEPOT Inc. (Barker, TX, USA), Gibson-BRL (Grand Island, NY, USA), and WElGENE Inc. (Daegu, Korea). Dimethyl sulfoxide (DMSO), MTT reagents, and Hoechst-33342 dye were obtained from Sigma-Aldrich (St. Louis, MO, USA). Invitrogen (Carlsbad, CA, USA) provided propidium iodide (PI) dye. Green/ROX QRTPCR Master Mix was obtained from Thermo Scientific (Foster, CA, USA).

The human HCC cell line, SNU449, was acquired from the Korean Cell Line Bank (Seoul, South Korea). Immortalized normal hepatocytes (MIHA) were provided by Dr. Roy-Chowdhury (Albert Einstein College of Medicine, Bronx, NY, USA). MIHA cells were cultured in Dulbecco’s modified Eagle’s medium (GenDEPOT, Barker, TX, USA), while SNU449 cells were cultured in RPMI1640 (GenDEPOT). Both mediums were supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, USA) and 100 U·mL⁻¹ penicillin-streptomycin (GenDEPOT). Cells were grown in a humidified incubator at 37 °C and 5% CO₂.

LP cells were isolated using a slightly modified previous method [13 ]. Briefly, jejunum tissues were cut into 0.5 cm pieces and washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) containing 1 mM DL-dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA), 30 mM ethylene-diamine-tetra acetic acid (EDTA; Thermo Fisher Scientific, Waltham, MA, USA), and 10 mM 4-[2-hydroxyethyl]-1-piperazineerhanesulfonic acid (HEPES; Thermo Fisher Scientific, Waltham, WA, USA) at 37 °C for 10 min (predigestion first step). Then, tissue samples were washed again in PBS containing 30 mM EDTA and 10 mM HEPES at 37 °C for 10 min (predigestion second step). After the washing step, tissues were transferred to 5 mL of 10% fetal bovine serum (FBS) containing RPMI 1640 (GenDEPOT, Barker, TX, USA) and inverted for 2 min (neutralization step). Lastly, the tissues were digested in 10% FBS containing RPMI 1640 with 0.5 mg/mL collagenase VIII (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h (digestion step). After the digestion step, isolated cells were applied to Percoll (GE Healthcare, Chicago, IL, USA) gradient centrifugation (40% Percoll on the top, 70% Percoll on the bottom).

Human fibrosarcoma cell line, HT1080, was adopted from KCLB (Korea Cell Line Bank, Jongro, Seoul, Korea), and cultured in T-75 flasks (SPL, Pocheon, Gyeonggi, Korea) in 5% CO₂ and at 37 °C in a humidified incubating condition, and the medium used was RPMI 1640 (GenDEPOT, Baker, TX, USA) with 10% fetal bovine serum (FBS) (GenDEPOT, Baker, TX, USA) and 100 unit/mL penicillin–streptomycin (Gibco-BRL, Grand Island, NY, USA). The cell lines were washed with PBS buffer (Gibco-BRL, Grand Island, NY, USA) and the medium was changed six times a week.
For cell viability assay, cell lines were transferred to 96-well plates at 5 × 10³ cells/well density. After their transfer, cells were cultured for 24 h and their medium was changed with fresh medium, which was followed by the treatment with samples (100, 50 and 10 µM). Cells were re-supplied with fresh medium after 24 h of incubation and treated with 100 µL of MTT solution (1 mg/mL), and further incubated for 4 h. Finally, the wells were aspirated and introduced into 100 µL of dimethyl sulfoxide (DMSO), in order to solubilize the formazan crystals for the measurement by a Victor3 reader (PerkinElmer, Waltham, MA, USA) at 540 nm optical density.