William s medium

Manufactured by Thermo Fisher Scientific

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William's medium is a laboratory culture medium used for the growth and maintenance of a variety of cell types, including mammalian cells. It provides a balanced, nutrient-rich environment to support cellular function and proliferation.

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William’s E medium (265 citations) William’s E medium with Glutamax (3 citations) William’s Medium E (WME) (2 citations) William’s E + GlutaMAX-I medium (2 citations)

24 protocols using william s medium

Cultivation and Differentiation of Liver Cells

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Plates and flasks were collagenized prior to cell seeding. NTCP-expressing HepG2-cells and RLR-expressing Huh7.5 cells were cultivated in D-MEM (Pan-Biotech, Aidenbach, Germany) supplied with 10% FCS, 2 mM L-Glutamine, 100 Units/mL Penicillin, 10 µg/mL Streptomycin, amino acids, sodium pyruvate and 30 µg/mL blasticidin as a selection marker. Prior to infection, cells were differentiated for 14 days by adding 2.5% DMSO (Sigma, St. Louis, MO, USA) to the growth medium. HepaRG-cells were cultivated in Williams medium (Invitrogen, Carlsbad, CA, USA) supplied with 10% non-heat-inactivated FCS Fetalclone II (HyClone, Thermo Scientific, Waltham, MA, USA) 2 mM L-Glutamine, 100 Units/mL Penicillin, 10 µg/mL Streptomycin, 0.023 IE/mL human insulin, 4.7 µg/mL hydrocortisone and 80 µg/mL Gentamicin. Cells were differentiated in 1.8% DMSO for 14 days prior to infection. Prestimulation of HepG2-NTCP cells was achieved using Polyinosinic:polycytidylic acid (Poly I:C) at a final concentration of 100 ng/mL.

Altstetter S.M., Quitt O., Pinci F., Hornung V., Lucko A.M., Wisskirchen K., Jung S, & Protzer U. (2021). Hepatitis-D Virus Infection Is Not Impaired by Innate Immunity but Increases Cytotoxic T-Cell Activity. Cells, 10(11), 3253.

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Antibody Reagents for Cell Signaling

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High-glucose Dulbecco’s modified Eagle medium, Williams medium, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA). Anti–mono-dimethyl arginine antibody (ab412) was obtained from Abcam, Inc (Cambridge, MA). Anti–alcohol dehydrogenase (ADH) was a gift from Dr Michael Felder (University of South Carolina, Columbia, SC). Cytochrome P450 2E1 (CYP2E1) (AB1252) was from EMD Millipore (Temecula, CA). Antibody to the STAT-1 (sc-592) and β-actin (sc-47778) antibodies were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). JMJD6 antibody (TA306835) and small interfering RNA (siRNA) (SR308094) were from OriGene (Rockville, MD); JMJD6 plasmid (plasmid 31358) was from Addgene (Cambridge, MA). Polymerase chain reaction (PCR) reagents, probes, and primers were from Life Technologies, Inc (Carlsbad, CA). Other reagents, all of analytical-grade quality, were from Sigma (St. Louis, MO).

Ganesan M., Tikhanovich I., Vangimalla S.S., Dagur R.S., Wang W., Poluektova L.I., Sun Y., Mercer D.F., Tuma D., Weinman S.A., Kharbanda K.K, & Osna N.A. (2017). Demethylase JMJD6 as a New Regulator of Interferon Signaling: Effects of HCV and Ethanol Metabolism. Cellular and Molecular Gastroenterology and Hepatology, 5(2), 101-112.

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Culturing and Inducing EMT in Cholangiocyte Cell Lines

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The non-malignant human cholangiocyte cell line MMNK-1 were obtained from the JCRB Cell Bank (Osaka, Japan) and cultured as recommended by the supplier. The CCA cell lines HuCCT1, HuH28 and OZ were obtained from the JCRB Cell Bank, and TFK-1 and RBE cells were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT (Tsukuba, Japan) [37 (link)]. HuCCT1, HuH28, TFK-1 and RBE cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Thermo Fisher Scientific). OZ cells were cultured in Williams’ medium (Thermo Fisher Scientific) with 10% FBS and 1% penicillin-streptomycin. MMNK-1 cells were cultured in DMEM high-glucose medium (Thermo Fisher Scientific) with 5% FBS and 1% penicillin-streptomycin. All cells were cultured at 37° C in a 5% CO₂ incubator. For all EV studies, EV-depleted medium was prepared using Exosome-Depleted FBS (Thermo Fisher Scientific). TGF-β was obtained from EMD Millipore Corporation (Billerica, MA, USA). Cells were treated with 10 ng/ml TGF-β for 72 h to induce EMT.

Ota Y., Takahashi K., Otake S., Tamaki Y., Okada M., Aso K., Makino Y., Fujii S., Ota T, & Haneda M. (2018). Extracellular vesicle-encapsulated miR-30e suppresses cholangiocarcinoma cell invasion and migration via inhibiting epithelial-mesenchymal transition. Oncotarget, 9(23), 16400-16417.

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Cell Line Maintenance and EMT Induction

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HuCCT1, HuH28, and OZ human CCA cell lines were obtained from the Japanese Cancer Research Resources Bank (JCRB) Cell Bank (Osaka, Japan), and TFK‐1 and RBE were purchased from the RIKEN BRC through the National Bio‐Resource Project of the MEXT (Tsukuba, Ibaraki, Japan) [14 (link)]. MMNK‐1 nonmalignant human cholangiocytes were obtained from the JCRB. HuCCT1, HuH28, TFK‐1, and RBE cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Thermo Fisher Scientific). OZ cells were cultured in Williams' medium (Thermo Fisher Scientific) containing 10% FBS and 1% penicillin–streptomycin. MMNK‐1 cells were cultured in Dulbecco's’ modified Eagle's medium (DMEM) high‐glucose medium (Thermo Fisher Scientific) with 5% FBS and 1% penicillin–streptomycin. All cells were cultured at 37 °C with 5% CO₂. For all EV studies, EV‐depleted medium was prepared using exosome‐depleted FBS (Thermo Fisher Scientific). Transforming growth factor (TGF)‐β was obtained from EMD Millipore Corporation (Billerica, MA, USA), and the cells were treated with 10 ng·mL⁻¹ TGF‐β for 72 h to induce the EMT.

Ota Y., Takahashi K., Otake S., Tamaki Y., Okada M., Yan I., Aso K., Fujii S., Patel T, & Haneda M. (2021). Extracellular RNA transfer from non‐malignant human cholangiocytes can promote cholangiocarcinoma growth. FEBS Open Bio, 11(12), 3276-3292.

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GBC Cell Lines NOZ and SGC-996 Culture

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The human GBC cell lines NOZ and SGC-996 were both purchased from Shanghai Institute Biological Science, Chinese Academy of Science (Shanghai, China). NOZ cells were cultured in William’s medium (Gibco, New York, USA) and SGC-996 cell line was cultured in Rosewell Park Memorial Institute 1640 (RPMI-1640) (Gibco). Both of the above media were supplemented with 10% fetal bovine serum (Gibco), 100 μg/ml streptomycin, and 100 U/ml penicillin (Hyclone, Logan, Uttah, USA). The two cell lines were maintained at 37°C in a humidified atmosphere with 5% CO₂.

Le Zou T., Wang H.F., Ren T., Shao Z.Y., Yuan R.Y., Gao Y., Zhang Y.J., Wang X.A, & Liu Y.B. (2019). Osthole inhibits the progression of human gallbladder cancer cells through JAK/STAT3 signal pathway both in vitro and in vivo. Anti-Cancer Drugs, 30(10), 1022-1030.

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Hepatocyte Isolation and TET3 Knockdown

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Mouse primary hepatocytes were prepared as previously described (Zhang et al., 2018 (link)). Cells were maintained in complete culture medium (Williams’ Medium [GIBCO,12551] supplemented with 5% FBS, 10 mM HEPES buffer [GIBCO, 15630–080], 2 mM L-Glutamine [GIBCO, 25030–081], 1% SPA [GIBCO, 15240–062], 4 mg/L insulin [GIBCO, 12585–014] and 1 μM dexamethasone [Sigma, D4902]). For TET3 knockdown experiments, cells seeded in 12-well plates were infected with AAV8-scr or AAV8-siTET3 at 6,000 gc/cell at 3 h after seeding. Medium was changed the next day, and cells were stimulated with TGF-β1 at a final concentration of 5 ng/ml in insulin- and FBS-free culture medium, followed by RNA and protein extraction 48 h later.
Human primary hepatocytes were obtained through the Liver Tissue Cell Distribution System, which was funded by NIH contract N01-DK-7–0004/HHSN267200700004C. Cells were maintained in complete culture medium (HMM medium [LONZA, CC-3197] supplemented with 1% SPA [GIBCO, 15240–062], 0.1 μM insulin [GIBCO, 12585–014] and 0.1 μM dexamethasone [Sigma, D4902]). To induce hepatocyte stress, the hepatocytes were incubated with glycochenodeoxycholic acid (GCDCA) (Sigma-Aldrich, G0759) at a final concentration of 100 μM for 48 h, followed by protein extraction.

Xu Y., Sun X., Zhang R., Cao T., Cai S.Y., Boyer J.L., Zhang X., Li D, & Huang Y. (2020). A Positive Feedback Loop of TET3 and TGF-β1 Promotes Liver Fibrosis. Cell reports, 30(5), 1310-1318.e5.

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Galllbladder Cancer Cell Line Cultivation

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The human gallbladder cancer cell lines GBC-SD, NOZ, SGC-996, OCUG, EHGB-1 and EHGB-2 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (CAS, Shanghai, China). The GBC-SD cells were grown in high-glucose DMEM (Gibco, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin(Hyclone, Logan, UT, USA). The NOZ cells were grown in William’s medium (Gibco) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin (Hyclone). The SGC cells were grown in 1640 medium (Gibco) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin (Hyclone), The OCUG, EHGB-1 and EHGB-2 cells were grown in high-glucose DMEM (Gibco) supplemented with 15 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin (Hyclone). All cells lines were maintained at 37 °C in a humidified atmosphere with 5 % CO₂. The A66 used in the in vitro and in vivo experiments was purchased from Selleck Chemicals (Houston, USA). Before conducting the CO-IP experiments, the GBC-SD and NOZ cells were starved for 12 h and 1 ng/mL EGF (Sangon Biotech, Shanghai, China) was added to the cell culture medium. The cells were maintained for 12 h to eliminate other confounding growth factor signals.

Zhao S., Cao Y., Liu S.B., Wang X.A., Bao R.F., Shu Y.J., Hu Y.P., Zhang Y.J., Jiang L., Zhang F., Liang H.B., Li H.F., Ma Q., Xu Y., Wang Z., Zhang Y.C., Chen L., Zhou J, & Liu Y.B. (2016). The E545K mutation of PIK3CA promotes gallbladder carcinoma progression through enhanced binding to EGFR. Journal of Experimental & Clinical Cancer Research : CR, 35, 97.

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Culturing and Treating HepG2-ERα Cells

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HepG2-ERα cells were obtained from Dr. David Shapiro (University of Illinois) and grown as previously described [14 (link)]. Cells were maintained in William’s medium (Gibco, Waltham, MA, USA) with phenol red and non-essential amino acid salts (NEAA), supplemented with 5% serum + II (HyClone, Logan, UT, USA), 100 µg/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), and 2.5% sodium bicarbonate. Cells were grown for at least 5 days in this media prior to use. William’s medium without phenol red was used for treating HepG2-ERα cells. Oleic acid (OA) used in the cell assays was purchased from Sigma Aldrich and it was dissolved in a small amount of DMSO and brought to the desired concentration by adding ethanol.

Zuo Q., Chen K.L., Arredondo Eve A., Liu Y.J., Kim S.H., Katzenellenbogen B.S., Katzenellenbogen J.A, & Madak-Erdogan Z. (2021). Pathway Preferential Estrogens Prevent Hepatosteatosis Due to Ovariectomy and High-Fat Diets. Nutrients, 13(10), 3334.

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Cultivation of Human Gallbladder Cancer Cell Lines

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The human gallbladder cancer cell lines GBC-SD and NOZ were purchased from the Shanghai Cell Institute Country Cell Bank. GBC-SD cells were cultured in high-glucose Dulbecco’s modified eagle’s medium (DMEM) (Gibco, California, USA), and NOZ cells were cultured in William’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 μg/mL streptomycin, and 100 U/mL penicillin (Hyclone, Logan, UT) at 37°C in an atmosphere containing 5.0% CO₂. Cells were routinely grown in 100-mm plastic tissue culture dishes (Corning, New York, USA) and harvested using a trypsin-EDTA solution when they reached the logarithmic growth phase. Cells were maintained in these culture conditions for all experiments.

Shu Y.J., Weng H., Bao R.F., Wu X.S., Ding Q., Cao Y., Wang X.A., Zhang F., Xiang S.S., Li H.F., Li M.L., Mu J.S., Wu W.G, & Liu Y.B. (2014). Clinical and prognostic significance of preoperative plasma hyperfibrinogenemia in gallbladder cancer patients following surgical resection: a retrospective and in vitro study. BMC Cancer, 14, 566.

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Establishing Human Gallbladder Cancer Cell Lines

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The human GBC cell lines NOZ, EH-GB-1, EH-GB-2, SGC-996 and GBC-SD were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The OCUG-1 cell line was obtained from the Health Science Research Resources Bank (Osaka, Japan). The NOZ cell line was maintained in William’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). The GBC-SD cells were maintained in DMEM medium (Gibco). The EH-GB-1, EH-GB-2, SGC-996 and OCUG-1 cells were cultured in RPMI 1640 (Gibco). DHA was purchased from Selleck Chemicals and dissolved in DMSO. Primary antibodies against TCTP, vinculin, paxillin and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA).

Zhang F., Ma Q., Xu Z., Liang H., Li H., Ye Y., Xiang S., Zhang Y., Jiang L., Hu Y., Wang Z., Wang X., Zhang Y., Gong W, & Liu Y. (2017). Dihydroartemisinin inhibits TCTP-dependent metastasis in gallbladder cancer. Journal of Experimental & Clinical Cancer Research : CR, 36, 68.

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