Mkn45

Manufactured by American Type Culture Collection

Sourced in United States, China, Japan

The MKN45 is a laboratory equipment product offered by the American Type Culture Collection. It is a cell culture device designed for the maintenance and propagation of cell lines.

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MKN45 cells (3 citations)

356 protocols using mkn45

Host Cell Response to H. pylori

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Human gastric epithelial cells AGS and MKN-45 (CRL-1739; ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Gibco, CA, USA) containing 10% fetal bovine serum (Sigma Aldrich, MO, USA) and 1% penicillin/streptomycin (Gibco) at 37 °C in 5% CO₂ atmosphere. AGS cells were transiently transfected with the YAP CDNA plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
When the cells reached 70% confluence, they were serum-starved overnight prior to H. pylori infection. All H. pylori strains were grown in Brucella broth supplemented with 5% fetal bovine serum at 37 °C for 24 h under microaerobic conditions; OD_600nm of bacterial suspensions was then adjusted with 1% FBS DMEM media to concentrations corresponding to a multiplicity of infection (MOI; the number of bacteria per cell at the onset of infection) of 50, 100 and 200. After co-culture with H. pylori strains for 6 or 24 h, AGS cells were collected for qPCR, Western blotting, or immunofluorescent staining.

Li N., Feng Y., Hu Y., He C., Xie C., Ouyang Y., Artim S.C., Huang D., Zhu Y., Luo Z., Ge Z, & Lu N. (2018). Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway. Journal of Experimental & Clinical Cancer Research : CR, 37, 280.

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Cell Line Characterization and Manipulation

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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).

Tanaka T., Kutomi G., Kajiwara T., Kukita K., Kochin V., Kanaseki T., Tsukahara T., Hirohashi Y., Torigoe T., Okamoto Y., Hirata K., Sato N, & Tamura Y. (2016). Cancer-associated oxidoreductase ERO1-α drives the production of VEGF via oxidative protein folding and regulating the mRNA level. British Journal of Cancer, 114(11), 1227-1234.

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Gastric Cancer Cell Culture and Patient-Derived Samples

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Human gastric epithelial cells GES-1 (Bogu Biotech, Shanghai, People’s Republic of China) and human GC cell lines CTC-141 and MKN45 (ATCC, Manassas, VA, USA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; HyClone, Logan, UT, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS) at 37°C with 5% CO₂.
The 83 pairs of GC tissue samples and adjacent normal tissue samples were collected from Zhoupu Hospital during 2013–2016. Written informed consent was obtained from each patient, and the experiments involving human tissue samples were approved by the Clinical Research Ethics Committee of Zhoupu Hospital.

Wei S., Zhong L., Wang X, & Zhang W. (2017). Low expression of GATA3 promotes cell proliferation and metastasis in gastric cancer. Cancer Management and Research, 9, 769-780.

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Gastric Cancer Cell Line Culture

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Gastric cancer cell lines (SGC-7901, MKN-45, AGS, MKN-28, and MGC-803) and human normal gastric mucosal epithelial cells (NGEC) were purchased from ATCC (American Type Culture Collection, ATCC). NGEC, AGS cells were cultured in 90% DMEM (Biological Industries, Cat. No. 06-1055-57-1A), 10% FBS (Biological Industries, Cat. No. 04-007-1A), and 1% penicillin/streptomycin. SGC-7901, MKN-45, MKN-28, and MGC-803 cells were cultured in 90% RPMI (Biological Industries, Cat. No. 01-101-1A), 10% FBS, and 1% penicillin/streptomycin. Although MGC-803 cell reports show cross-contamination with HeLa, there are still many research groups using MGC-803 as a model for studying gastric cancer cells (Zhai et al., 2019 (link); Li et al., 2020 (link); Peng et al., 2020 (link)).

Shen X., Zhao K., Xu L., Cheng G., Zhu J., Gan L., Wu Y, & Zhuang Z. (2021). YTHDF2 Inhibits Gastric Cancer Cell Growth by Regulating FOXC2 Signaling Pathway. Frontiers in Genetics, 11, 592042.

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Cell Lines for Gastric Cancer Research

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GES-1 is a SV40 transformed human fetal gastric epithelial cell line, which were established in 1994 and proved to maintain a normal cytoskeleton, positive in PAS reaction and were non-tumourigenic in nude mice.30 (link) GES-1, SGC7901, MKN28, MKN45 and AGS cells (originally purchased from ATCC) were maintained in RPMI-1640 medium; BGC823 and HEK293T cells (originally purchased from ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Thermo Scientific HyClone, Beijing, China).

Li T., Guo H., Li H., Jiang Y., Zhuang K., Lei C., Wu J., Zhou H., Zhu R., Zhao X., Lu Y., Shi C., Nie Y., Wu K., Yuan Z., Fan D.M, & Shi Y. (2019). MicroRNA-92a-1–5p increases CDX2 by targeting FOXD1 in bile acids-induced gastric intestinal metaplasia. Gut, 68(10), 1751-1763.

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Cell Culture of Gastric Cell Lines

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The human gastric mucosal epithelial cell line (GES-1), GC cell lines (AGS, NCI-N87, MKN-45, SNU-1, and SNU-16), and HEK293T cells were purchased from ATCC and authenticated by STR profiling. These cells were cultured in F12K, RPMI 1640 or DMEM (Gibco, USA) medium with 10% FBS (Gibco) and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ atmosphere.

Liu J., Niu L., Hao J., Yao Y., Yan M, & Li H. (2023). circIPO7 dissociates caprin-1 from ribosomes and inhibits gastric cancer cell proliferation by suppressing EGFR and mTOR. Oncogene, 42(13), 980-993.

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Immunofluorescence Staining of Co-cultured Cells

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Human foreskin-derived fibroblasts (BJ) and human gastric adenocarcinoma cell lines, AGS and MKN-45, were purchased from ATCC (USA). BJ and AGS cells were maintained in DMEM GlutaMAX (Invitrogen, ThermoFisher Scientific, Germany) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. MKN-45 cells were maintained in RPMI 1640 medium (Invitrogen, ThermoFisher Scientific, Germany) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
BJ cells (1×10⁴) and human gastric adenocarcinoma cells (AGS and MKN-45) were separately seeded in 24-wells plates with one 22×22 mm coverslip per well. After cultured for 12 hours, one coverslip with BJ cells and another coverslip with either AGS or MKN-45 were transferred into a single well of a 6-wells plate and maintained in 2 ml of DMEM complete medium for 48 hours. Cells on each coverslip were fixed with 4% paraformaldehyde and prepared for immunofluorescence staining. All pictures of immunofluorescence staining were taken at 63x magnification under an oil objective.

Gao Y., Yin S.P., Xie X.S., Xu D.D, & Du W.D. (2017). The relationship between stromal cell derived SPARC in human gastric cancer tissue and its clinicopathologic significance. Oncotarget, 8(49), 86240-86252.

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Characterization of Gastric Cancer Cell Lines

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Gastric cancer cell lines were purchased from ATCC (KATOIII, NCI-N87, SNU-16), KCLB (SNU-668, SNU-601, SNU-638), JCRB (MKN-45, NUGC-4) and ECACC (HGC-27). Microsatellite Instability (MSI) status was previously assessed for all cell lines and found negative for all but one cell line (SNU-638). Identity of each cell line was determined through independent karyotyping. Cells were checked for mycoplasma contamination. Cells were cultured in their recommended media conditions at 37°C. Afterward, the cells were processed into suspensions with standard procedures. Briefly, this process involved trypsinizing the cells, followed by inactivation by fetal bovine serum (FBS). We performed washes by centrifugation at 400 g in 1× phosphate-buffered saline with 0.04% bovine serum albumin. To remove cellular debris and cellular aggregates, we filtered cells through a Flowmi cell strainer (Wayne, NJ, USA) before proceeding to single-cell DNA and RNA sequencing.

Andor N., Lau B.T., Catalanotti C., Sathe A., Kubit M., Chen J., Blaj C., Cherry A., Bangs C.D., Grimes S.M., Suarez C.J, & Ji H.P. (2020). Joint single cell DNA-seq and RNA-seq of gastric cancer cell lines reveals rules of in vitro evolution. NAR Genomics and Bioinformatics, 2(2), lqaa016.

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Establishment of Herceptin-resistant GC Cell Lines

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Two human HER2-positive GC cell lines (NCI-N87 and MKN45) were purchased from ATCC. All cell lines were cultured in RPMI 1640 (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL; Invitrogen) and incubated at 37°C in a humidified chamber containing 5% CO₂.
Herceptin resistant GC cell lines (NCI-N87-HR and MKN45-HR) were established as described previously.18 Briefly, the parental NCI-N87 and MKN45 cells were treated with increasing doses of Herceptin gradually for 6 months until Herceptin resistance was stably acquired. NCI-N87-HR and MKN45-HR cells were normally maintained with 10 µg/mL Herceptin.

Gao X., Lu C., Chen C., Sun K., Liang Q., Shuai J., Wang X, & Xu Y. (2020). ARPP-19 Mediates Herceptin Resistance via Regulation of CD44 in Gastric Cancer. OncoTargets and therapy, 13, 6629-6643.

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Gastric Cancer Cell Line Profiling

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A total of forty pairs of GC specimens and control specimens were acquired from GC patients who underwent surgery at The Fourth Hospital of China Medical University (Liaoning, Shengyang). Cell lines (HGC-27, MKN-45, SGC-7901, MGC-803 and GES cells) were acquired from ATCC, USA and cultured in DMEM (Gibco, BRL, UK) supplemented with streptomycin/penicillin and FBS (Gibco, BRL, UK). miR-142-5p mimic, pcDNA-MSC-AS1, siRNA-DDX5 and their controls (20 nM) were obtained from Shanghai GenePharma. Cell transfections were carried out with a Lipofectamine kit (Invitrogen, CA, USA).

Liu Y., Li L., Wu X., Qi H., Gao Y., Li Y, & Chen D. (2021). MSC-AS1 induced cell growth and inflammatory mediators secretion through sponging miR-142-5p/DDX5 in gastric carcinoma. Aging (Albany NY), 13(7), 10387-10395.

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