WM115, RPMI7951, SKMEL24, WM793, SKMEL28, SKMEL5, A375, SKMEL3, and MALM3M human melanoma cell lines were purchased from ATCC. 501MEL and WM9 cell lines were kindly provided by Dr Robert Ballotti and M4BE cell line by Dr Thibault Voeltzel (Centre Léon Bérard). All these BRAFV600 human melanoma cell lines were cultured in DMEM complemented with 10% FBS (Cambrex) and 100 U/ml penicillin–streptomycin (Invitrogen). In order to authenticate the cell lines, the expected major genetic alterations were verified by NGS sequencing. The absence of mycoplasma contamination was checked every 3 weeks with the MycoAlert detection kit (Lonza). Patient‐derived short‐term cultures (< 10) were established from BRAFV600 metastatic melanomas, before treatment for GLO and C‐09.10, or after acquisition of resistance to vemurafenib for ESP and GOKA. C‐09.10 were kindly provided by Dr Robert Ballotti (Nice). These short‐term cell cultures were grown in RPMI complemented with 10% FBS and 100 U/ml penicillin–streptomycin. PLX4032/vemurafenib and GDC0973/cobimetinib were purchased from Selleck Chemicals (Houston, TX, USA) and reconstituted in DMSO. Generation of A375‐R and SKMEL5‐R resistant models was performed by treating cells chronically with increasing doses of PLX4032 for 2–3 months. All BRAFi‐resistant cell lines were permanently cultured in the presence of 3 μM PLX4032.
Wm793
Manufactured by American Type Culture Collection
Sourced in United States
The WM793 is a cell culture incubator designed for controlled temperature and humidity environments. It features precise temperature and CO2 control to support the growth and maintenance of various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Sk mel 28, by American Type Culture Collection (4 mentions)
Wm115, by American Type Culture Collection (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Rpmi media, by Thermo Fisher Scientific (2 mentions)
Plx4032, by Selleck Chemicals (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sk mel 2, by American Type Culture Collection (2 mentions)
Mst2 smartpool sirna, by Horizon Discovery (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Lox imvi, by American Type Culture Collection (2 mentions)
Hs294t, by American Type Culture Collection (2 mentions)
Plx4032 vemurafenib, by Selleck Chemicals (1 mentions)
Sk mel 5, by American Type Culture Collection (1 mentions)
Sk mel 3, by American Type Culture Collection (1 mentions)
Gdc0973 cobimetinib, by Selleck Chemicals (1 mentions)
Rpmi 7951, by American Type Culture Collection (1 mentions)
Sk mel 24, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Mycoalert detection kit, by Lonza (1 mentions)
12 protocols using wm793
1
Establishment and Authentication of Melanoma Cell Lines
2
Melanoma Cell Line Culturing Protocol
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The SK-Mel147, WM 1366, and SK-Mel173 melanoma cell lines were generously provided by M.S. Soengas (CNIO, Madrid, Spain). The MeWo, SK-Mel131, M17, and TRP melanoma cell lines were used in a previous study [41 (link)]. The WM 1552C, WM 35, WM 793, and WM 115 melanoma cell lines were purchased from ATCC. Cells were cultured in DMEM:F12 medium (1:1) supplemented with 10% fetal bovine serum (Lonza) and maintained at 37 °C in a humidified incubator with 5% CO2. The analyses were performed within a few passages after thawing. Cells were routinely tested for Mycoplasma contamination.
3
Establishment and Cultivation of Melanoma Cell Lines
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Human melanoma cell line WM793 (Coriell, catalog no. WC00062, RRID: CVCL_8787) was purchased from the ATCC. Murine BrafV600E melanoma cells established from a C57BL/6 Braf+/LSL-V600E;Tyr::CreERT2+/o;p16INK4a-/− mouse was generously provided by Dr. S. Zelenay (The University of Manchester, Manchester, England; refs. 15, 21 (link)). Cell lines were cultivated in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell cultures were maintained in 5% CO2 at 37°C. All the cell lines had been authenticated using short tandem repeat DNA fingerprinting using the AmpFLSTR Identifiler kit, within the last 3 years. All experiments were performed with Mycoplasma-free cell lines.
4
Culturing Human and Mouse Melanoma Cell Lines
5
Comprehensive BRAF and MAPK Signaling Assays
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A375, SK-Mel28, WM-793, and SK-Mel2 were obtained from ATCC and were validated by sequencing before initiation of the study. All cells were grown in RPMI media (Gibco) supplemented with 10% foetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). Media for the resistant cells contains 2 μM PLX4032 (Selleck chemicals). HeLa cells were also validated by sequencing and grown in DMEM (Gibco) 10% foetal bovine serum and 2 mM L-glutamine. The cells were maintained at 37°C and 5% CO2. Cells were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol and the amount of DNA and siRNA is indicated in each experiment. FLAG-BRAF, -BRAFR509H, -BRAF V600E/R509H, and BRAFV600E, MYC-LATS1, and FLAG-MST2 have been described (Romano et al, 2014a (link); Jambrina et al, 2016 (link); Quinn et al, 2021 (link)) HA-ubiquitin was a gift from Edward Yeh (plasmid # 18712; Addgene) (Kamitani et al, 1997 (link)). MST2 SMARTpool siRNA is from Dharmacon (M-012200) and was validated before (Matallanas et al, 2007 (link)). Bortezomib (S1013), trametinib (S2673), dabrafenib (GSK2118436), encorafenib (LGX818), sorafenib tosylate (S1014), and TAK-632 are from Selleck Chemicals and Proteasome inhibitor I from Calbiochem (539160).
6
Melanoma Cell Lines and Samples
7
Cell Culture and Transfection Protocols
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Cell culture and transfection: A375, SK-Mel28, WM-793 and SK-Mel2 were obtained from ATCC and were validated by sequencing before initiation of the study. All cells were grown in RPMI media (Gibco, USA) supplemented with 10% foetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). Media for the resistant cells contains 2 μM PLX4032 (Selleck chemicals, USA). HeLa cells were also validated by sequencing and grown in DMEM (Gibco) 10% foetal bovine serum and 2 mM L-glutamine. The cells were maintained at 37°C and 5% CO 2 . Cells were transfected with Lipofectamine 2000 (Invitrogen, USA) following manufacturers protocol and the amount of DNA and siRNA is indicated in each experiment. FLAG-BRAF, -BRAF R509H , -BRAF V600E/R509H , and BRAF V600E , MYC-LATS1 and FLAG-MST2 have been described (Romano et al., 2014a , Quinn et al., 2021 , Jambrina et al., 2016) HA-Ubiquitin was a gift from Edward Yeh (Addgene plasmid # 18712) (Kamitani et al., 1997) . MST2 SMARTpool siRNA is from Dharmacon (M-012200, CO, USA) and was validated before (Matallanas et al., 2007) . Bortezomib (S1013), trametinib (S2673) are from Selleck Chemicals and Proteasome inhibitor I from Calbiochem (539160, CA, USA).
8
Characterization of Human Melanoma Cell Lines
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Human melanoma cell lines WM35, WM793, 451LU, A2058 and A375 were purchased from American Type Culture Collection (ATCC) in 2014. Experiments were performed on cells passaged for no more than 6 months. WM35 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, CA). WM793 and 451LU were maintained in MCDB153 medium with 2% fetal bovine serum. A2058 and A375 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. All these melanoma cell lines were authenticated by short-tandem repeat (STR) fingerprinting by Beijing Microread Genetics Company Limited in 2016 and tested for mycoplasma contamination. Normal human melanocytes that were isolated from human foreskin specimens obtained during circumcision surgery were cultured in Medium 254 (Invitrogen, Carlsbad, CA) supplemented with Human Melanocyte Growth Supplement (Gibco). All the cells were maintained at 37 °C with 5% CO2.
9
Culturing Melanoma Cell Lines
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Human primary melanoma cell lines (WM35, WM115, and WM793) and metastatic melanoma cell lines (A375, 1205Lu, and HS294T) were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (Thermo Scientific, Rockford, IL, USA) supplemented with 10% fetal bovine serum (Gemini Bioproducts, West Sacramento, CA, USA), 100 IU/ml penicillin-100 μg/ml streptomycin (Mediatech, Manassas, VA, USA) at 37°C and 5% CO2 in the incubator. Melanoma-conditioned media (MCM) was obtained from culture supernatants of human melanoma cells after 24 h of cultivation in OptiMEM (Life Technologies, Grand Island, NY, USA) and centrifuged at 210 × g for 5 min (26 (link)). These cell lines have been authenticated using the short tandem repeat (STR) fingerprinting by the Barbara Davis Center Bioresource Core at the University of Colorado Anschutz Medical Campus. Cells were regularly monitored for mycoplasma contamination using PCR.
10
Melanoma Cell Line Analysis
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The human melanoma cell lines A375, MeWo, LOX IMVI, and WM793 were obtained from the American Type Culture Collection (ATCC), and WM 266.4 was provided by Dr. Michael Davies (MD Anderson). Cells were cultured in Dulbecco’s modified Eagle medium containing 5% fetal bovine serum at 37°C and in 5% CO2 and 95% air. For DETA NONOate treatment, cells were cultured to 75% confluency, and freshly prepared DETA NONOate stock was added to the final concentration as indicated in the text. After treatment times, cells were washed twice with cold phosphate-buffered saline and were harvested for analysis of pAKTS473 levels, AKT kinase activity, AKT substrate phosphorylation, and PTEN activity. An iNOS expressing construct was established as described by us previously (10 (link)). Cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s specifications.
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