Medium 254

Manufactured by Thermo Fisher Scientific

Sourced in United States, Israel, China, Germany

The Medium 254 is a laboratory equipment designed for the preparation and storage of cell culture media. It is suitable for a variety of cell types and applications in the life sciences industry.

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Growth medium M-254 (6 citations) HMGS supplemented melanocytes growth medium-254 (2 citations)

121 protocols using medium 254

Culturing Mouse Melanoma and Human Melanocytes

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B16F10 cells (Cascade Biologics, Portland, OR, USA), a mouse melanoma cell line, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/ streptomycin) in a humidified 5% CO₂ atmosphere at 37 °C. Cultured human epidermal melanocytes (neonatal, moderately pigmented donor) were obtained from Cascade Biologics (Portland, OR, USA) and maintained in Medium 254 (Cascade Biologics) supplemented with Human Melanocyte Growth Supplement (HMGS) at 37 °C in a humidified atmosphere containing 95% air/5% CO₂. For experiments, melanocytes were used at passage 2 or 5 and maintained in Medium 254 (Cascade Biologics) supplemented with HMGS.

Lee S.E., Park S.H., Oh S.W., Yoo J.A., Kwon K., Park S.J., Kim J., Lee H.S., Cho J.Y, & Lee J. (2018). Beauvericin inhibits melanogenesis by regulating cAMP/PKA/CREB and LXR-α/p38 MAPK–mediated pathways. Scientific Reports, 8, 14958.

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Melanocyte Isolation and Characterization

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Primary normal human melanocytes were isolated from human foreskin specimens obtained during circumcision surgery. The primary melanocytes were grown in Medium 254 (Cascade Biologics, Portland, OR, USA) containing Human Melanocyte Growth Supplement (Cascade Biologics). The primary melanocytes were used between 2^nd and 4^th passage in all experiments. The immortalized normal human epidermal melanocyte cell line PIG1 and a vitiligo melanocyte cell line PIG3V (both gifts from Dr. Caroline Le Poole, Loyola University Chicago, Maywood, IL, USA) was cultured in Medium 254 (Cascade Biologics) containing Human Melanocyte Growth Supplement (Cascade Biologics) and 5% fetal bovine serum (Invitrogen, San Diego, CA, USA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. H₂O₂ (Sigma-Aldrich, St Louis, MO, USA) was used at concentration of 500 μM. Rapamycin (Abcam, Cambridge, UK) was used at concentration of 400 nM. Chloroquine (Sigma-Aldrich) was used at concentration of 50 μM. All subjects consented by written and informed agreement for inclusion in this study. All experiment protocols were approved by the Ethics Committee of the Fourth Military Medical University, in accordance with the Declaration of Helsinki principles.

He Y., Li S., Zhang W., Dai W., Cui T., Wang G., Gao T, & Li C. (2017). Dysregulated autophagy increased melanocyte sensitivity to H2O2-induced oxidative stress in vitiligo. Scientific Reports, 7, 42394.

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Culturing Murine Melanoma and Human Melanocytes

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B16F10 murine melanoma cells (B16 cells) were purchased from Riken Cell Bank (Tsukuba, Japan) and maintained as a monolayer culture in DMEM supplemented with 10% FBS, 4 mmol/L L-glutamine, 50 units/mL penicillin, and 50 µg/mL streptomycin, and incubated in a 37 °C humidified atmosphere with 5% CO₂. Human epidermal melanocytes (HEMs) were purchased from Gibco Invitrogen cell culture and maintained in Medium 254 (Carlsbard, CA, USA) supplemented with human melanocyte growth supplement (HMGS) (s) were purchased from Gibco Invitrogen cell culture and maintained in Medium 254 (Carlsbard, MA, USA), which contains bovine pituitary extract, fetal bovine serum, bovine insulin, bovine transferrin, basic fibroblast growth factor, hydrocortisone, heparin, and phorbol 12-myristate 13-acetate (PMA), following the supplier’s instructions. During treatment with samples, HGMS without PMA (HMGS-2) was used. Photographs of the cells were taken using a Leica DFC290 HD camera (Beckman Coulter, Brea, CA, USA).

Villareal M.O., Chaochaiphat T., Makbal R., Gadhi C, & Isoda H. (2022). Molecular Analysis of the Melanogenesis Inhibitory Effect of Saponins-Rich Fraction of Argania spinosa Leaves Extract. Molecules, 27(19), 6762.

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Quantitative Real-Time PCR Assay

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Cell culture materials Medium 254, Medium 254 supplement, fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and antibiotics were obtained from Gibco (Waltham, MA, USA). Short interfering RNA (siRNA) transfection reagent, Trizol reagent, 1-bromo-3-chloropropane (BCP), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO, USA). ToolScript MMLV RT kit and TOOLS 2× SYBR quantitative real-time polymerase chain reaction (qPCR) Mix was purchased from Biotools (Taipei, Taiwan).

Li P.H., Liu L.H., Chang C.C., Gao R., Leung C.H., Ma D.L, & David Wang H.M. (2018). Silencing Stem Cell Factor Gene in Fibroblasts to Regulate Paracrine Factor Productions and Enhance c-Kit Expression in Melanocytes on Melanogenesis. International Journal of Molecular Sciences, 19(5), 1475.

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Culturing Human Melanoma Cell Lines

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Human melanoma cell lines (WM35, WM902B, WM983A, WM983B, 1205Lu, WM793, and A375) harboring the BRAF V600E mutation and human melanoma cell line (WM852) with wildtype BRAF were obtained from Dr. Meenard Herlyn (The Wistar Institute). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) media supplemented with fetal bovine serum (FBS) (10%), L-glutamine (2 mM), penicillin (1%), and streptomycin (1%). Human primary melanocytes (Life Technologies) were grown in Medium 254 (Gibco) with human melanocyte growth supplements (Gibco). Cell lines were incubated at 37°C in 5% CO₂.

Lee H.J., Chen Z., Collard M., Chen F., Chen J.G., Wu M., Alani R.M, & Cheng J.X. (2021). Multimodal Metabolic Imaging Reveals Pigment Reduction and Lipid Accumulation in Metastatic Melanoma. BME Frontiers, 2021, 9860123.

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Inducing ER Stress in Human Epidermal Melanocytes

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Human primary epidermal melanocytes were acquired from American Type Culture Collection. Cells were cultured in Medium 254 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with human melanocyte growth supplement (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C and 5% CO₂.
To induce ER stress, human primary epidermal melanocytes (1x10⁶ cells per well) were treated with 3 µM tunicamycin (TM; Sigma-Aldrich; Merck KGaA) (22 (link)) at 37˚C for 24, 48 and 72 h.
Primary epidermal melanocytes were transfected with 1 µg control plasmid (cat no. sc-437275; Santa Cruz Biotechnology, Inc.) or 1 µg RIPK1 plasmid (cat no. sc-422681-ACT; Santa Cruz Biotechnology, Inc.) for 24 h using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. Reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis were used to detect the efficiency of cell transfection. 24 h after cell transfection, subsequent experiments were performed.

Sun X., Wang T., Huang B., Ruan G, & Xu A. (2020). RIPK1 regulates the survival of human melanocytes upon endoplasmic reticulum stress. Experimental and Therapeutic Medicine, 19(5), 3239-3246.

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Culturing Melanocyte and Melanoma Cell Lines

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Human normal melanocyte cell line (PIG1) and vitiligo melanocyte cell line (PIG3V) were kindly donated by Dr. Chunying Li (Xijing Hospital, Fourth Military Medical University, Xi'an, China). Cells were cultured in Medium 254 (Gibco, Grand Island, NY, USA) supplemented with Human Melanocyte Growth Supplement (Gibco), 5% fetal bovine serum (Gibco), and 1% penicillin-streptomycin antibiotic mix (Invitrogen, CAL, USA) at 37 °C with 5% CO_2.²⁸ (link) The B16 melanoma cell lines were cultured in Dulbecco's modified Eagle's medium (HyClone Laboratories, UT, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin antibiotic mix at 37 °C in a 5% CO₂ atmosphere.

Zou D.P., Chen Y.M., Zhang L.Z., Yuan X.H., Zhang Y.J., Inggawati A., Kieu Nguyet P.T., Gao T.W, & Chen J. (2020). SFRP5 inhibits melanin synthesis of melanocytes in vitiligo by suppressing the Wnt/β-catenin signaling. Genes & Diseases, 8(5), 677-688.

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Melanocyte Oxidative Stress Model

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The immortalized human normal melanocyte cell line PIG1 (purchased from Bena Culture Collection, Beijing, China) was cultured in Medium 254 (Gibco, Grand Island, NY) supplemented with Human Melanocyte Growth Supplement (Gibco) and 5% fetal bovine serum at 37 °C with 5% CO₂. We established an oxidative stress model in the PIG1 cells by treating them with 1.0 mM H₂O₂ (Sigma-Aldrich, USA) for 24 h. We added 10 μM MeCbl (Sigma-Aldrich) 48 h before the H₂O₂ treatment as needed. In addition, we treated PIG1 cells with 1.5 µM ML385 (MedChemExpress, USA), a compound that inhibits the activation of Nrf2, to further explore the MeCbl mechanisms.

An R., Li D., Dong Y., She Q., Zhou T., Nie X., Pan R, & Deng Y. (2021). Methylcobalamin Protects Melanocytes from H2O2-Induced Oxidative Stress by Activating the Nrf2/HO-1 Pathway. Drug Design, Development and Therapy, 15, 4837-4848.

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Chemical Agents for Melanocyte and Keratinocyte Assays

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BCI-215, a chemical produced from BCI by medicinal chemistry [36 (link),37 (link),38 (link)], was purchased from MCE (MedChemExpress, Monmouth Junction, NJ, USA) and prepared as a solution in DMSO. Kojic acid, cholera toxin (CT), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), and fetal bovine serum (FBS) were purchased from WelGENE (Daegu, South Korea). Medium 254, EpiLife™ Medium, Human Melanocyte Growth Supplement (HMGS), Human Keratinocyte Growth Supplement (HKGS), Antibiotic-Antimycotic (AA), and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). Medium 254 (Gibco) was obtained from Cascade Biologics (Portland, OR, USA).

Lee J.H., Choi M.E., An H., Moon J.W., Yeo H.J., Song Y, & Chang S.E. (2022). BCI-215, a Dual-Specificity Phosphatase Inhibitor, Reduces UVB-Induced Pigmentation in Human Skin by Activating Mitogen-Activated Protein Kinase Pathways. Molecules, 27(17), 5449.

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Murine Melanocyte Cell Culture Protocol

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Melan-a melanocytes are normal, immortalized murine melanocyte cells derived from C57BL/6 mice. Melan-a melanocytes were obtained from Dorothy Bennett (St. George's Hospital, London, UK). Melan-a melanocytes were maintained in a RPMI-1640 (Welgene, Korea) medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO). Melan-a Leaden (or Ashen) melanocytes are immortalized Melanophilin (or Rab27a) mutant murine melanocyte cells derived from C57BL/6J 54 . Leaden and Ashen melanocytes were obtained from Dorothy Bennett (St. George's Hospital, London, UK). Cells were maintained in RPMI-1640 (Welgene, Korea) medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and 200 nM PMA and 2 nM cholera toxin (Sigma-Aldrich, St. Louis, MO). HDFn cells were maintained in Dulbecco's Modified Eagle Medium (Welgene, Korea) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. COS7 monkey kidney cells were maintained in SMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. NHEM cells were maintained in a Medium 254 (gibco) supplemented with 1%HMGS, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Jo C.S., Park H.I., Jung H.J., Park J.I., Lee J.E., Myung C.H, & Hwang J.S. (2020). A novel function of Prohibitin on melanosome transport in melanocytes. Theranostics, 10(9), 3880-3891.

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