Twenty seven established cutaneous melanoma cell lines were used in this study. Twenty three early-passage (<20) melanoma lines were established from AJCC stage III and IV melanoma patients who received elective surgery at JWCI and were authenticated with autologous peripheral blood leukocytes. Stage I/II melanoma lines (WC00060, WC00080, WC00081 and WC00062) were obtained from Coriell Institute (Camden, NJ) and have been tested by short tandem repeat DNA profiles. Cells were cultured in a humidified chamber with 5% CO2 at 37°C in either RPMI-1640 (Corning, Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gemini Bio-Products, Sacramento, CA) and 1% penicillin-streptomycin, or MCDB-153 (Sigma-Aldrich, St. Louis, MO) / Leibovitz’s L-15 (Life Technologies, Carlsbad, CA) supplemented with 2% FBS, Insulin (5 μg/ml, Sigma-Aldrich), and 1% penicillin-streptomycin (9 (link),17 (link)). All the cell line experiments were completed within 12 passages or <4 months from thawing.
Mcdb153
Manufactured by Merck Group
Sourced in United States, Germany
MCDB153 is a laboratory instrument designed for cell culture applications. It maintains optimal environmental conditions for cell growth and development. The core function of MCDB153 is to provide a controlled and consistent environment for in vitro cell cultivation.
Automatically generated - may contain errors
Lab products found in correlation
Insulin, by Merck Group (10 mentions)
Trypsin edta, by Thermo Fisher Scientific (9 mentions)
Cacl2, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Leibovitz s l 15, by Thermo Fisher Scientific (4 mentions)
Bovine insulin, by Merck Group (3 mentions)
Leibovitz s l 15, by GE Healthcare (3 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Puregene dna isolation kit, by Qiagen (2 mentions)
Methylcap kit with high salt elution, by Diagenode (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Leibovitz s l 15, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Calcium chloride, by Merck Group (2 mentions)
T75 flasks, by Merck Group (2 mentions)
Leibovitz l 15, by Thermo Fisher Scientific (2 mentions)
32 protocols using mcdb153
1
Melanoma Cell Line Characterization
2
Human Melanocyte and Melanoma Cell Culture
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The human melanocyte cell line was acquired from Rio de Janeiro Cell Bank (code BCRJ 0190) and was grown in DMEMF12 (Invitrogen, Scotland, UK) at pH 7.2 supplemented with 20% fetal bovine serum (Invitrogen, Scotland, UK), 2.5 mM glutamine (Invitrogen, Scotland, UK), 500 μM sodium pyruvate (Invitrogen, Scotland, UK), 1.4 μM hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 1 nM Triiodotreonin (T3) (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL Insulin (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL Transferrin (Sigma-Aldrich, St. Louis, MO, USA) and 10 ng/mL Epidermal Growth Factor (Sigma-Aldrich, St. Louis, MO, USA). The patient-derived melanoma cells lines WM1552C, WM793, WM1366, WM983B and Lu1205 were kindly provided by Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA, USA) and grown in TU medium (80% MCDB153 (Sigma-Aldrich, St. Louis, MO, USA medium plus 20% Leibovitz medium (Invitrogen, Scotland, UK) supplemented with 2% FBS and 1.68 mM CaCl2 (https://wistar.org/our-scientists/meenhard-herlyn , accessed on 1 April 2022). WM155C lineage corresponds to radial growth phase (RGP) melanoma cells, WM793 and WM1366 lineages correspond to vertical growth (VGP) melanoma cells and WM983B and Lu1250 correspond to the metastatic melanoma cells. The cultures were kept in an incubator at 37 °C in a humidified atmosphere containing 5% CO2.
3
Melanoma cell expression analysis
4
Methylation Profiling of Melanoma Cells
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Example 1
Methyl-binding domain (MBD)-sequencing was performed on six melanoma cell lines (WM35, WM3248, WM164, A375, M14, SK-MEL-28) and normal human epidermal melanocytes (NHEM) provided by Dr. Léon van Kempen (McGill University, Montreal, Canada). Authentication of all cell lines was performed using short tandem repeat (STR) profiling (DSMZ, Braunschweig, Germany). WM cell lines were cultured in W489 medium consisting of four parts of MCDB153 (Sigma-Aldrich, Zwijndrecht, The Netherlands) and one part of L15 (Sigma-Aldrich, Zwijndrecht, The Netherlands), A375, M14, and SK-MEL-28 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Breda, the Netherlands). Cells were supplemented with 2% or 10% heat inactivated fetal calf serum (Hyclone Perbio Science, Erembodegem-Aalst, Belgium), respectively. NREM cells were cultured in ready-to-use medium supplied by Promocell (Heidelberg, Germany). Genomic DNA was isolated using the PUREGENE® DNA isolation kit (Gentra systems, Minneapolis, Minn.) according to the manufacturer's instructions.
Genomic DNA of all samples was subjected to methylation-enrichment sequencing using the MethylCap kit with high-salt elution (Diagenode, Liege, Belgium) as described previously [25]. For each sample, and each methylation core, the maximum read count was used in downstream analyses.
5
Murine Melanoma Cell Culture and Characterization
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Murine melanoma B2905, derived from a UV-irradiated HGF-transgenic mouse on a C57BL/6 background (Patel et al., 2017 (link)) and B16F10.9 (Porgador et al., 1991 (link)) were used. The B2905 cell line was grown in RPMI (Biological Industries) containing 10% heat inactivated FBS (GIBCO), 1% L-glutamine (Biological Industries), 1% Penicillin/Streptomycin antibiotics (Invitrogen) and 12.5mM HEPES (Sigma) buffer. B16F10.9 cells were grown in DMEM medium (GIBCO) containing 10% heat inactivated FBS, 1% L-glutamine, 1% Penicillin/Streptomycin antibiotics. Both cell lines originated from male mice. Primary cell lines that were generated from solid tumors, derived from UVB-B2905 as described below, were grown in Tu2% media [80% MCDB 153 (Sigma), 20% Leibovitz’s L-15 (Sigma), 5 μg/mL bovine insulin (Sigma), 2% FBS (GIBCO), and 1.68 mM CaCl2]. The murine lymphoma mutant cell line RMA-s (Ljunggren et al., 1990 (link)) were cultured in RPMI medium supplemented with 10% heat-inactivated FBS, 40 μg/ml gentamycin sulfate and 5x10−5M β-mercaptoethanol. All cells were cultured using standard procedures in a 37°C humidified incubator with 5% CO2. Cells were tested routinely for Mycoplasma using Mycoplasma EZ-PCR test kit (cat#20-700-20, Biological Industries).
6
Reconstructed Human Epidermal Equivalent
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Epidermal model is a reconstructed human epidermis equivalent developed by the Advanced Skin Research and Bioengineering Department at Ashland Global Skin Research Center, Sophia Antipolis, France (Capallere et al. 2018). To obtain the RHE, normal human primary keratinocytes (NHK) were isolated from skin explants from a healthy 2-year old donor after surgery. Briefly, the biopsy sample was incubated overnight at 4 °C in Dispase II (Sigma -Aldrich, Saint Quentin Falavier, France) to remove epidermis from dermis, the remaining cells were then dissociated by Trypsin (Sigma -Aldrich, Saint Quentin Falavier, France), centrifugated and resuspended in culture medium (MCDB 153 modified (Sigma-Aldrich, Saint Quentin Falavier, France). To reconstruct RHEs, the isolated NHKs and cultured KiPS-40 were seeded on a 0.5 cm2 inert polycarbonate membrane (Nunc, Denmark) in a chemically defined medium and were air-lifted for several days at 37 °C under 5% CO2.
7
Characterizing Transformed Mouse Fibroblasts
8
Culturing Metastatic Melanoma Cell Lines
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The human melanoma cell lines 501mel, HMB2 and Mel Im were derived from metastases of malignant melanomas. Cells were maintained in DMEM supplemented with penicillin (400 U/ml), streptomycin (50 μg/ml), L-glutamine (300 μg/ml) and 10 % fetal calf serum (FCS) and split at a 1:5 ratio every three days. The cells were cultured under a humidified atmosphere of 8 % CO2 at 37°C. Further human melanoma cell lines were a kind gift by Meenhard Herlyn (Wistar Institute, Philadelphia, PA) and maintained in a culture medium consisting of MCDB153 (Sigma Aldrich) with 20 % Leibovitz's L-15 (PAA Laboratories, Coelbe, Germany), 2 % FCS (PAA Laboratories), 1.68 mM CaCl2 (Sigma Aldrich) and 5mg/ml insulin (Sigma Aldrich). Tissue origins of metastatic melanoma cell lines were the lymph nodes (WM239a, WM9, WM1158, and WM1232) and lungs (451Lu and 1205Lu). Primary melanocytes (NHEM) were isolated from human foreskins and purchased from PromoCell (Heidelberg, Germany) and cultured in Growth Medium M2 (C-24300).
Recombinant NRN1 was purchased from Sigma-Aldrich. Human NRN1 was expressed in E.coli and was diluted in PBS buffer containing 0.1 % BSA as a carrier protein as was used in a concentration of 50 ng/ml.
Recombinant NRN1 was purchased from Sigma-Aldrich. Human NRN1 was expressed in E.coli and was diluted in PBS buffer containing 0.1 % BSA as a carrier protein as was used in a concentration of 50 ng/ml.
10
Culturing Melanoma Cell Lines as Monolayers and Spheroids
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