The human melanoma A375 cell line was obtained from ATCC. Cells were grown in DMEM (Mediatech Inc.) and were supplemented with 10% fetal bovine serum (FBS), 1 µg/mL hydrocortisone, and 1% penicillin and streptomycin. The murine melanoma B78-D14 (B78) cell line is derived from B16 melanoma as previously described [13 (link)] and was obtained from Ralph Reisfeld (Scripps Research Institute) in 2002. B78 cells were grown in RPMI-1640 w/L-glutamine (Mediatech Inc.) and were supplemented with 10% FBS, 100 U/mL penicillin, and 100ug/mL streptomycin. Cell line authentication was performed per ATCC guidelines using morphology, growth curves, and Mycoplasma testing within 6 months of use. All chemicals were purchased from Sigma. Cell culture media and supplements were obtained from Life Technologies, Inc.
A375 cell line
Manufactured by American Type Culture Collection
Sourced in United States, Italy
The A375 cell line is a human malignant melanoma cell line. It is derived from the skin of a 54-year-old Caucasian female. The A375 cell line is commonly used in research for the study of melanoma.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Cremophor rh40, by Merck Group (2 mentions)
A375 cell line, by European Collection of Authenticated Cell Cultures (2 mentions)
Trametinib, by MedChemExpress (2 mentions)
Peg400, by Merck Group (2 mentions)
Labrafil m 1944 cs, by Gattefosse (2 mentions)
Cell culture media and supplements, by Thermo Fisher Scientific (1 mentions)
Dabrafenib, by Selleck Chemicals (1 mentions)
A375 melanoma cell line, by American Type Culture Collection (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Selumetinib, by Selleck Chemicals (1 mentions)
32 protocols using a375 cell line
1
Culturing Human and Murine Melanoma Cell Lines
2
Evaluating MAPK and AKT Pathways in A375 Melanoma Cells
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A375 cell line was obtained from ATCC (LGC Standards, Italy). This cell line derived from a 54 year old female with malignant melanoma and represents a good model for studying the role of MAPK and AKT pathways because it is affected by the single alteration displayed in the B-RAF gene (V600E) (See Cosmic web site, http://cancer.sanger.ac.uk/cosmic ). In fact, the presence of other genetic alterations, such as the mutations in KRAS or NRAS genes, may determine the activation of MAPK pathway.
The A375 melanoma cell line, obtained from ATCC (LGC Standards, Italy), were cultured in a humidified 5% CO2 incubator at 37°C with RPMI-1640 supplemented with 2 mmol/L L-glutamine, 100 IU penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS), purchased from LONZA (Walkersville, USA). The A375 were plated in 70 mm cell-culture dishes at density of 500.000 cells and after 24h were treated for 48h with Dabrafenib (Selleck Chemical, USA) to final concentration of 2, 1, 0.5, 0.25 and 0.125 nM. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, USA) was used as control.
The A375 melanoma cell line, obtained from ATCC (LGC Standards, Italy), were cultured in a humidified 5% CO2 incubator at 37°C with RPMI-1640 supplemented with 2 mmol/L L-glutamine, 100 IU penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS), purchased from LONZA (Walkersville, USA). The A375 were plated in 70 mm cell-culture dishes at density of 500.000 cells and after 24h were treated for 48h with Dabrafenib (Selleck Chemical, USA) to final concentration of 2, 1, 0.5, 0.25 and 0.125 nM. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, USA) was used as control.
3
Culturing A375 Melanoma Cells
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A375 cell line (ATCC, Manassas, VA, USA; catalog number: CRL-1619) has been verified by STR profiling and tested negative for mycoplasma contamination. Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (AusGeneX, Molendinar, Qld, Australia) and 1% penicillin-streptomycin (P/S; Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere in a 5% CO2 incubator at 37°C.
4
Establishment and Characterization of PF130 Cell Line
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The PF130 cell line was established from a malignant pleural effusion. Briefly, the sample was centrifuged at 1200× g for 10 min, then the supernatant was removed. The pellet was resuspended in RPMI1640 containing 10% FBS, and 100 U/mL penicillin–streptomycin and the suspension was seeded on a 25 cm2 tissue culture flask. The adherent cells were cultured for 10 passages to obtain a tumor cell culture free of non-malignant cells before experiments were initiated. The study was approved by the Ethics Committee at the University Hospital Essen (#18-8208-BO) and written consent from the patient was obtained. The A375 cell line was purchased from ATCC. Both cell lines were subjected to Single Nucleotide Analysis by Multiplex Cell Line Authentication (Multiplexion, Heidelberg, Germany). Both cell lines were cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 in a humidified atmosphere. Selumetinib and trametinib were purchased from Selleck Chemicals (Houston, TX, USA), and were dissolved in DMSO at 10 mM concentration and stored at −80 °C in aliquots. MAP855 was obtained from Novartis and dissolved in DMSO.
5
Cell Line Authentication and Maintenance
6
Culture of A375 Melanoma Cells
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The A375 cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany) and 1% penicillin/streptomycin mixture (Pen/Strep, 10,000 IU/mL; PromoCell, Heidelberg, Germany), as previously described [40 (link)]. Melanoma cells were passaged at confluence after treatment with 5 mM Ethylenediaminetetraacetic acid (EDTA).
7
Metastatic Melanoma Patient Blood Analysis
8
Culturing Mouse and Human Cell Lines
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Normal embryonic mouse fibroblasts (NIH 3T3) (kindly obtained from Asst Prof. Dr Fahsai Kantawong, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand) and the human malignant melanoma (A375) cell line (ATCC, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, New York, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 µg/mL), and maintained in a humidified atmosphere of 5% CO2 at 37°C.
9
S100B Protein Expression and Purification
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Reagents. Cloning vector pMD18-T vector was purchased from TransGen Biotech (Beijing, China). A375 cell line was obtained from the American Type Culture Collection (Manassas, VA, uSA). NdeI, XhoI, and T4 DNA ligases were purchased from New England Biolabs, Ltd. (Beijing, China). Isopropyl-β-d-thiogalactopyranoside (IPTG) was purchased from Merck & Co., Inc. (Darmstadt, Germany). qIA quick gel extraction and nucleotide removal kits were obtained from qiagen China Co., Ltd. (Shanghai, China). Commercial recombinant human S100B protein and S100B antibody were from Abcam (Cambridge, MA, uSA). unless otherwise stated, other reagents and materials were performed as described in the literature (7) . Animals were purchased from the Experimental Animal Room of the Institute of Health and Environmental Medicine (Tianjin, China). This study was approved by the Committee of the Institute of Health and Environmental Medicine.
10
Sera Collection and Cell Line Maintenance
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Human sera from 8 healthy donors and 8 MM patients were obtained between February 2008 and December 2013, from the Department of Pathology and Environmental Health Center for Asbestos, Pusan National University Hospital (Yangsan, Korea) subsequent to diagnosis and staging. Human MM NCI-H226, NCI-H2452, NCI-H28 and MSTO-211H cell lines, human lung cancer A-427 and A549 cell lines, and the human melanoma cancer A375 cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). Human colon cancer SNU-C1, SNU-C2A, SNU-C4 and SNU-C5 cell lines, human ovarian cancer SNU-8 and SNU-840 cell lines were obtained from the Korean Collection for Type Cultures (Jeongeup, Korea). All cell lines were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum medium (Gibco; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin. Cells were cultured at 37°C in a humidified 5% CO2 atmosphere. The present study was performed with approval from the Ethical Committee of Pusan National University Yangsan Hospital (Yangsan, Korea).
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