The human breast cancer lines MDA-MB-157, MDA-MB-231, MDA-MB-468 and MCF7, the human pancreas cancer cell line MIAPaCa2 and the human gastric cancer cell line MKN45 were purchased from ATCC (Manassas, VA, USA). MDA-MB-157 cells were cultured in Roswell Park Memorial Institute-1640 (Sigma-Aldrich, St Louis, MO, USA) and MDA-MB-231, MDA-MB-468, MCF7, MIAPaCa2 and MKN45 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) at 37 °C in 5% CO2. Short-hairpin RNA for human ERO1-α (TR313168) was purchased from OriGene (Rockville, MD, USA) and transfected to MDA-MB-231, MCF7 and MIAPaCa2 cells using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). To establish cells with ERO1-α-overexpression, MDA-MB-231 cells were transfected with human ERO1-α cDNA using Lipofectamine 2000 (Life Technologies) as per the manufacturer's instructions. Cells were stably propagated under puromycin selection (1 or 2 μg ml−l). The ERO1-α inhibitor EN460 was purchased from Millipore (Billerica, MA, USA).
Mkn 45
Manufactured by Merck Group
Sourced in United States
The MKN-45 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose tool for various laboratory applications. The core function of the MKN-45 is to provide a reliable and consistent platform for conducting research and analysis activities.
Automatically generated - may contain errors
Lab products found in correlation
Mkn45, by American Type Culture Collection (3 mentions)
Cisplatin, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Mcf 7, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Mia paca 2, by Merck Group (1 mentions)
Mda mb 157, by Merck Group (1 mentions)
Mda mb 468, by Merck Group (1 mentions)
Short hairpin rna for human ero1 α tr313168, by OriGene (1 mentions)
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
En460, by Merck Group (1 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Mcf 7, by Merck Group (1 mentions)
Mda mb 468, by American Type Culture Collection (1 mentions)
Mda mb 231, by Merck Group (1 mentions)
Mia paca 2, by Thermo Fisher Scientific (1 mentions)
Mda mb 157, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
7 protocols using mkn 45
1
Cell Line Characterization and Manipulation
2
Generating Cisplatin-resistant Gastric Cancer Cells
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Human normal gastric mucosa cells (GES-1) and GC cells (N87, HGC-27, MKN-45, and AGS) were acquired from Procell (Wuhan, China). All cells were cultured in RPMI1640 (Procell) added with 1% penicillin/streptomycin (Procell) and 10% FBS (Procell) at 37°C and 5% CO2.
The DDP-resistant GC cells (MKN-45/DDP and AGS/DDP) were generated by continuous gradient exposing MKN-45 and AGS cells to increasing doses of DDP (Sigma-Aldrich, St. Louis, MO, United States) from 0.03 μg/ml until the cells acquired resistance to 1 μg/ml (0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6 0.7, 0.8, 0.9, 1 μg/ml) for about 12 months. The DDP-resistant cells were cultured in cisplatin (1 μM)-contained RPMI1640 (Procell) and cultured in cisplatin-free RPMI1640 (Procell) for 1 week before further use.
The DDP-resistant GC cells (MKN-45/DDP and AGS/DDP) were generated by continuous gradient exposing MKN-45 and AGS cells to increasing doses of DDP (Sigma-Aldrich, St. Louis, MO, United States) from 0.03 μg/ml until the cells acquired resistance to 1 μg/ml (0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6 0.7, 0.8, 0.9, 1 μg/ml) for about 12 months. The DDP-resistant cells were cultured in cisplatin (1 μM)-contained RPMI1640 (Procell) and cultured in cisplatin-free RPMI1640 (Procell) for 1 week before further use.
3
Isolation and Culture of Gastric Cancer Cells
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This study was carried out in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of the University of Tokyo (Permit No:10034). The written informed consent was obtained from each patient. Intraperitoneal free cells were obtained from peritoneal lavages or ascites recovered from patients who underwent abdominal surgery for gastric cancer or paracentesis. Informed written consent was obtained from all patients. After the centrifugation at 1500 rpm for 15 min, the pellets were resuspended in PBS+0.02% EDTA and overlaid on Ficoll-Hypaque solution (Pharmacia Biotech, Piscataway, NJ). After centrifugation at 3000 rpm for 10 min, the intermediate layer was taken and washed twice. These cells were cultured with DMEM media in Type I collagen-coated plates or flasks (IWAKI, Tokyo JAPAN). After reaching confluence, the cells were removed by treatment with 0.02% EDTA and trypsin, and passaged and cultured for up to 3 weeks.
The human gastric cancer cell line MKN45 was obtained from Riken (Tukuba JAPAN) [13] (link), and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO), 100 units/ml penicillin and 100 mg/ml streptomycin (Life Technologies, Inc., Grand Island, NY).
The human gastric cancer cell line MKN45 was obtained from Riken (Tukuba JAPAN) [13] (link), and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO), 100 units/ml penicillin and 100 mg/ml streptomycin (Life Technologies, Inc., Grand Island, NY).
4
Establishment of Cisplatin-Resistant Gastric Cancer Cell Lines
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Human GC cell lines (MGC-803 and MKN-45) were brought from Bena Culture Collection (Beijing, China). Both cell lines were maintained in RPMI 1640 (HyClone, Logan, UT, USA) with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), penicillin (100 U/mL) and streptomycin (100 mg/mL) (Invitrogen, Carlsbad, CA, USA). Continuous exposure to stepwise-enhancing concentrations of cisplatin (Sigma-Aldrich, St. Louis, MO, USA) was used to establish cisplatin-resistant MGC-803 and MKN-45 cells. These cells were initially incubated in medium with a relatively low concentration of cisplatin (1 µM) in medium for 4 weeks. Subsequently, the surviving cells were exposed to a middle concentration of cisplatin (2 µM) for 6 weeks. Lastly, MGC-803 and MKN-45 cells were incubated in the culture medium containing a high concentration of cisplatin (5 µM). All cells were grown in a moist atmosphere with 5% carbon dioxide incubator at 37°C.
5
Cell Line Culturing Across Cancers
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Human GC cell lines (MKN1, MKN45, MKN74, and TMK1), CRC cell lines (Caco-2, SW48, LS174T, and HCT116), and BC cell lines (SK-BR-3 and BT-474) used in this study were all obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in media consisting of Roswell Park Memorial Institute medium 1640 (MKN1, MKN45, MKN74, TMK1, SW48, and LS174T), Dulbecco's modified Eagle medium (SK-BR-3, BT-474, and HCT116) or minimal essential medium (MEM) (Caco-2) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 2% L-glutamine (MP Biomedicals, Eschwege, Hesse, Germany), 2% pyruvate (Sigma-Aldrich), 1% MEM non-essential amino acid solution (Sigma-Aldrich), 1% antibiotic/antimyotic solution (Sigma-Aldrich), and 0.1% tylosin solution (Sigma-Aldrich). Cells were incubated at 37°C in an atmosphere containing 5% CO2.
6
Gastric Cancer Cell Line Cultivation
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Four GC cell lines (AGS, MKN-45, HGC-27, MKN-7) and one human normal gastric epithelial cell line (GES-1) were all available from ATCC (Manassas, VA) and preserved at 37 °C with 5% CO 2 . RPMI-1640 (Gibco BRL, Grand Island, NY) with the supplementation of with 10% FBS (Gibco) and 1% Pen/Strep solution was used for cell culture. The anticancer agent cisplatin used to treat HGC-27 and MKN-45 cells was available from Sigma-Aldrich (St. Louis, MI). Besides, 20 μg/mL of actinomycin D (ActD) and 40 μg/mL of cycloheximide (CHX) were also acquired from Sigma-Aldrich.
7
Cell Culture Maintenance of Human and Murine T Cells
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The acute human T cell leukemia line, Jurkat clone E6.1, and the murine cytotoxic T cell line, CTLL-2, from American Type Culture Collection (ATCC; Manassas, VA, USA) were maintained in RPMI-1640 culture medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (JRH Bioscience, Lenexa, KS, USA), 100 U/mL of penicillin, and 100 µg/mL of streptomycin (Sigma-Aldrich). The human gastric cancer cell line, MKN-45, with or without the luciferase gene, from Cell Bank (Riken BioResource Center, Ibaraki, Japan), was grown in DMEM (Sigma-Aldrich) with supplements, as described above. The MKN-45 cells were detached and harvested after treatment with trypsin-EDTA/PBS (Sigma-Aldrich) for 5 min. The cells were maintained at 37°C in humidified 5% CO2/air, and the culture medium was changed twice a week.
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