Human melanoma cell lines (SK-MEL-28, SK-MEL-3, A375, HT-144, and Hs294T) were purchased from the American Type Culture Collection (ATCC, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactive fetal bovine serum (FBS; Gibco/Thermo Fisher Scientific, USA) in a CO2 incubator at 37°C. Human epidermal melanocytes were purchased from Scien-Cell Research Laboratories (USA) and cultured in Melanocyte Medium (ScienCell Research Laboratories) containing 10% FBS. HEK293T cells were purchased from the Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (China) and cultured in DMEM with 10% FBS.
Melanocyte medium
Manufactured by ScienCell
Sourced in United States
Melanocyte medium is a specialized cell culture medium designed to support the growth and maintenance of human melanocytes, which are the pigment-producing cells found in the skin. The medium provides the necessary nutrients and growth factors required for the optimal proliferation and differentiation of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Ht144, by American Type Culture Collection (5 mentions)
A2058, by American Type Culture Collection (5 mentions)
Sk mel 28, by American Type Culture Collection (4 mentions)
Sk mel 1, by American Type Culture Collection (4 mentions)
Human epidermal melanocytes, by ScienCell (2 mentions)
Sk mel 3, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Hs294t, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Complete medium, by Thermo Fisher Scientific (1 mentions)
A2058, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection kit, by Thermo Fisher Scientific (1 mentions)
Wm115, by American Type Culture Collection (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
Rpmi 7951, by American Type Culture Collection (1 mentions)
10 protocols using melanocyte medium
1
Culturing Human Melanoma Cell Lines
2
Profiling Melanoma Tissues and Cells
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A total of 22 pairs of human primary melanoma tissues and adjacent nontumorous skin specimens were obtained from patients who received surgical resections in Heilongjiang Provincial Hospital (Harbin, P.R. China) from January 2015 to November 2016. None of these patients underwent chemotherapy or radiotherapy prior to surgery. The resected tissues were immediately snap frozen in liquid nitrogen and stored at −80°C in a cryogenic refrigerator prior to further use. This study was approved by the Ethics Committee of Heilongjiang Provincial Hospital. Written informed consent was obtained from all participants.
Human epidermal melanocytes (HEMs) obtained from ScienCell Research Laboratories, Inc. (San Diego, CA, USA) were grown in a melanocyte medium (ScienCell Research Laboratories, Inc.) under conditions recommended by the manufacturer. Five melanoma cell lines, namely, A375, A2058, HT144, SK-MEL-1, and SK-MEL-28, were purchased from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 100 mg/ml penicillin, and 100 mg/ml streptomycin (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All the cell lines were cultured at 37°C in a humidified incubator with 5% CO2.
Human epidermal melanocytes (HEMs) obtained from ScienCell Research Laboratories, Inc. (San Diego, CA, USA) were grown in a melanocyte medium (ScienCell Research Laboratories, Inc.) under conditions recommended by the manufacturer. Five melanoma cell lines, namely, A375, A2058, HT144, SK-MEL-1, and SK-MEL-28, were purchased from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 100 mg/ml penicillin, and 100 mg/ml streptomycin (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All the cell lines were cultured at 37°C in a humidified incubator with 5% CO2.
3
Culturing Human Melanocytes and Cell Lines
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Human epidermal melanocyte (HEM) was bought from ScienCell (Carlsbad, CA, USA) and cultured in Melanocyte Medium (ScienCell). Melanoma cell lines A375 and A2058 were bought from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (fetal bovine serum (FBS); Invitrogen, Carlsbad, CA, USA) was used for culturing A375 and A2058 cells. Human umbilical vein endothelial cells (HUVECs) were bought from ATCC. Complete medium (Thermo Fisher Scientific) was used for culturing HUVECs. All the cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.
4
Jianbai Xiang Pig Melanocyte Isolation and Culture
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The laboratory successfully isolated, identified, cultured, and preserved third-generation Jianbai Xiang pig melanocytes and 293T cells. 293T cells were cultured with Dulbecco’s Modified Eagle Medium (Gibco, Beijing, China), and melanocytes were cultured in Melanocyte Medium (ScienCell Research Laboratories, USA), both supplemented with 10% fetal bovine (FBS, Gibco, USA) and 1% penicillin-streptomycin (P/S, Solarbio Life Sciences, Beijing, China). The cells were incubated at 37 °C and 5% CO2 in a humidified incubator.
Five pairs of siRNA sequences (Supplementary Table S4 ) were synthesized by Shanghai Jima Biotechnology Co., Ltd. based on the full sequence of the pig TYRP1 gene (GenBank No. 537,161). The siRNA with the highest silencing efficiency was selected and transfected into Jianbai Xiang pig melanocytes using the Lipofectamine™ 2000 transfection kit (Invitrogen, USA). The Dual luciferase reporter vectors (TYRP1-wt-F and TYRP1-wt-R) were constructed by Guizhou Hongdal Biotechnology Co., Ltd. (Supplementary Table S5 ), and miRNA-221-3p mimic, inhibitor, and negative control (NC) were constructed by Shanghai Gene Pharma Co., Jianbai Xiang pig melanocytes were transfection with ssc-miR-221-3p/NC by reached 60%~75% confluency, and 3 replicates were set up in each group.
Five pairs of siRNA sequences (Supplementary Table S
5
Human Melanocyte and Melanoma Cell Lines
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The experiments were performed in accordance with the Declaration of Helsinki guidelines. The study was approved by the Clinical Research Ethics Board of the University of British Columbia (Certificate H12-02653). With informed consent, biopsies were obtained and stored in RNAlater solution (Life Labs) as previously described [23 (link), 31 (link)] [32 (link)]. Biopsy tissues were archived and stored in −20°C in RNAlater solution (Invitrogen, Canada). Human epidermal melanocytes were purchased from ScienCell (Carlsbad, USA) and cultured in melanocyte medium (2201, ScienCell, Carlsbad, USA). Melanoma cell lines A375, RPMI 7951, SH4, WM-115, SK-MEL-1, SK-MEL-3, SK-MEL-24 were purchased from ATCC (Manassas, USA). Cells were cultured in growth medium as Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, USA) and 1X Antibiotic-Antimycotic (15240062, Gibco, Burlington, Canada) at 37°C in 5% CO2 humidified atmosphere.
6
Melanocyte Isolation and Cell Cycle Analysis
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Normal human melanocytes (NHM) were isolated from healthy skin tissues, and lgCMN cells were isolated from patients' tumor lesions, following previously described procedures [25 (link), 26 (link)]. Both NHM and lgCMN cells were cultured in melanocyte medium (ScienCell, Carlsbad, CA, USA) according to the manufacturer's instructions. The human melanoma cell line A375 was purchased from American Type Culture Collection (Manassas, VA, USA) and grown in RPMI-1640 medium containing 10% fetal bovine serum (Gibco, San Diego, CA, USA) and 1% antibiotic antimycotic solution (Gibco) at 37°C and 5% CO2. For cell cycle assays, A375 cells, P2 NHM, and P2 lgCMN cells were prepared in phosphate-buffered saline (PBS) at a density of 5 × 105 cells/mL. The cell suspensions were pipetted into 70% ethanol on ice and kept for two hours. After washing the cells with PBS, they were stained using Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China) for two hours. A flow cytometer, Beckman CytoflexS (Beckman Coulter, Brea, CA, USA), was used to evaluate the cell cycle.
7
Regulation of Melanoma Cell Behavior by miR-342 and ZEB1
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Human epidermal melanocytes (HEMs) were purchased from ScienCell Research Laboratories, Inc. (San Diego, CA, USA) and cultured in melanocyte medium (ScienCell Research Laboratories, Inc.) according to the manufacturer’s instructions. Four human melanoma cell lines (A375, A2058, SK-MEL-28, and HT144) were obtained from the American Type Culture Collection (Manassas, VA, USA). All melanoma cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained at 37°C in a humidified incubator with a mixture of 5% CO2.
For functional experiments, miR-342 mimic, miRNA negative control mimic (miR-NC), small interfering RNA (siRNA) targeting zinc-finger E-box-binding homeobox 1 (ZEB1 siRNA), and negative control siRNA (NC siRNA) were purchased from Guangzhou RiboBio Co., Ltd (Guangzhou, P.R. China). ZEB1 overexpression plasmid pCMV-ZEB1 and empty plasmid pCMV were synthesized by Guangzhou GeneCopoeia Co., Ltd (Guangzhou, P.R. China). Cells were plated into six-well plates at a density of 6 × 105 cells/well 24 h prior to transfection. Cell transfection was conducted using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer’s protocols.
For functional experiments, miR-342 mimic, miRNA negative control mimic (miR-NC), small interfering RNA (siRNA) targeting zinc-finger E-box-binding homeobox 1 (ZEB1 siRNA), and negative control siRNA (NC siRNA) were purchased from Guangzhou RiboBio Co., Ltd (Guangzhou, P.R. China). ZEB1 overexpression plasmid pCMV-ZEB1 and empty plasmid pCMV were synthesized by Guangzhou GeneCopoeia Co., Ltd (Guangzhou, P.R. China). Cells were plated into six-well plates at a density of 6 × 105 cells/well 24 h prior to transfection. Cell transfection was conducted using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer’s protocols.
8
Culturing Human Melanocytes and Melanoma Cells
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Human epidermal melanocytes (HEMs) were bought from ScienCell Research Laboratories, Inc. (San Diego, CA, USA) and grown in a melanocyte medium (ScienCell Research Laboratories, Inc.). Four human melanoma cell lines, ie, A375, A2058, SKMEL1, and HT144, were purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific), 100 U/mL penicillin, and 100 µg/mL streptomycin was utilized for culturing melanoma cells. All cell lines were maintained at 37°C in a humidified atmosphere supplied with 5% CO2.
9
Melanoma Tissue and Cell Line Samples
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Tissue samples and cell lines. This study was approved by the Ethics Committee of Subei People's Hospital of Jiangsu province. Written informed consent was provided by all of these patients participated in this research. From May 2014 to February 2016, a total of 21 pairs of melanoma tissues and corresponding adjacent normal tissues were gathered from melanoma patients who suffered a surgical resection at Subei People's Hospital of Jiangsu province. None patients had been treated with chemotherapy, radiotherapy or other treatment prior to surgery. All tissue samples were immediately frozen in liquid nitrogen and stored in -80˚C until further use.
Melanoma cell lines, including A375, A2058, HT144 and SK-MEL-28, were obtained from the American Type Culture Collection (Manassas, VA, USA), and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 mg/ml penicillin and 100 mg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Human epidermal melanocytes (HEM) were acquired from ScienCell Research Laboratories, Inc. (San Diego, CA, USA) and maintained in melanocyte medium (ScienCell Research Laboratories, Inc.) according to the manufacturer's protocol. All the cell lines were grown in a humid incubator at 37˚C with 5% CO 2 .
Melanoma cell lines, including A375, A2058, HT144 and SK-MEL-28, were obtained from the American Type Culture Collection (Manassas, VA, USA), and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 mg/ml penicillin and 100 mg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Human epidermal melanocytes (HEM) were acquired from ScienCell Research Laboratories, Inc. (San Diego, CA, USA) and maintained in melanocyte medium (ScienCell Research Laboratories, Inc.) according to the manufacturer's protocol. All the cell lines were grown in a humid incubator at 37˚C with 5% CO 2 .
10
Culturing Human Epidermal Melanocytes and Melanoma Cell Lines
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Human Epidermal Melanocytes (HEM) were purchased from Sciencell (USA) and cultured in Melanocyte Medium (Sciencell, USA) according to the manufacturer's instructions. Human melanoma cell lines SK-MEL-1 and A375 were obtained from American Type Culture Collection (Manassas, VA, USA). A375 and SK-MEL-1 cells were maintained in RPMI-1640 medium or DMEM medium respectively, supplemented with 10% fetal bovine serum, in a 37°C humidified atmosphere of 5% CO 2 .
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