The human keratinocytes were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS; Hyclone, USA), penicillin G (100 U/mL), and streptomycin (100 μg/mL) (Invitrogen, USA). Human umbilical vein endothelial cells (HUVECs) were grown on 0.1% gelatin-coated cell culture dishes in M199 medium (Welgene, Korea) supplemented with 20% (v/v) FBS, 3 ng/mL basic fibroblast growth factor (R&D Systems, USA), 5 U/mL of heparin (Sigma, USA), and a penicillin-streptomycin-amphotericin B mixture (100 U/mL potassium penicillin, 100 mg/mL streptomycin sulfate, and 250 ng/mL amphotericin B; Lonza, Belgium) as a complete medium. Cells were cultured at 37°C in a humidified incubator with 5% CO2. Cells were plated in 96-well tissue culture plates (2 × 104 cells per well). After 1 day, they were treated with various concentrations (25, 50, 100, and 200 μg/mL) of peptides. Melittin (Sigma, USA) was used as the positive control. After incubation for 24 h, the viability of the cells was assessed by the Cell Titer 96 AQueous One Solution Cell Proliferation Assay according to the manufacturer’s protocol (Promega, USA). The optical density at 490 nm was measured with a microplate reader (Beckman DTX 8800 Multi Detector, USA).
M199 medium
Manufactured by Welgene
M199 medium is a cell culture medium that provides nutrients and growth factors necessary for the maintenance and growth of a variety of cell types in vitro. It is a widely-used basal medium that supports the cultivation of a range of mammalian cells, including those derived from human, animal, and plant sources.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by R&D Systems (2 mentions)
Heparin, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dtx 8800 multi detector, by Beckman Coulter (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Melittin, by Merck Group (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Amphotericin b, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Streptomycin sulfate, by Lonza (1 mentions)
Auranofin, by Merck Group (1 mentions)
P38 sirna, by Cell Signaling Technology (1 mentions)
Anti gapdh, by AbFrontier (1 mentions)
Pkcδ sirna, by Santa Cruz Biotechnology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Quantikine human vegf immunoassay kit, by R&D Systems (1 mentions)
5 protocols using m199 medium
1
Cytotoxicity Evaluation of Peptides
2
Korean Ginseng Modulates Redox Balance
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Korean Red Ginseng powder was supplied by the Korea Ginseng and Tobacco Central Research Institute (Daejeon, Korea). M199 medium and fetal bovine serum were obtained from Welgene Inc. (Daegu, Korea). TRIzol reagent and Lipofectamine RNAiMAX were obtained from Invitrogen (Carlsbad, CA). Auranofin was purchased from Sigma Chemical (St. Louis, MO). Anti-Thioredoxin reductase 1 and anti-GAPDH were purchased from AbFrontier (Seoul, Korea). SB203580 and Rottlerin were supplied by Calbiochem (La Jolla, CA), p38 siRNA (#6564) was obtained from Cell Signaling Technology (Beverly, MA), and PKC-δ siRNA (SC-36253) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals and reagents were of analytical grade.
3
Isolation and Characterization of Gintonin
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Crude gintonin was isolated from P. ginseng as described previously [19] . Gintonin is a glycolipoprotein containing ginseng protein complexed with LPA [20] (link). Ginsenosides were purchased from the LKT Laboratories Inc. (St. Paul, MN, USA). VEGF, basic fibroblast growth factor, and Quantikine human VEGF immunoassay kit were purchased from R&D Systems (Minneapolis, MN, USA). M199 medium and 0.1% gelatin solution were purchased from WelGENE (Daegu-si, Korea). Matrigel (growth factor reduced) and collagen type 1 were purchased from BD Biosciences (Bedford, MA, USA). All other reagents used were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
Transfection and Gene Silencing in HEK-293 and HUVEC Cells
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Human embryonic kidney (HEK)-293 cells (American Type Culture Collection, USA) were maintained in Dulbecco’s modified Eagle’s medium (HyClone, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin according to the supplier’s recommendations. Human umbilical vein endothelial cells (HUVECs) were cultured in M199 medium (Welgene, South Korea) containing 20% FBS, 3 ng/ml human FGF-basic (Peprotech, USA), and antibiotics. Prior to transient transfection, cells were seeded in 6- or 12-well plates. The following day, 0.5–2 μg/well TRPC4β and PKD1 cDNA was transfected into cells using the transfection reagent FuGENE 6 (Roche Molecular Biochemicals, USA) for electrophysiological experiments, according to the manufacturer’s protocol. For molecular biology experiments, Lipofectamine 2000 (Invitrogen, USA) was used as the transfection reagent. All experiments were performed 20–30 hours after transfection. For gene silencing, siRNA was transfected using the transfection reagent RNAi Max (Invitrogen, USA) according to the manufacturer’s instructions. Control siRNA (Cat. SN-1003), human PKD1 siRNA (Cat. 1117253) and human TRPC4 (Cat. 1156755) were purchased from Bioneer (South Korea). Human STAT1 (Cat. sc-44123) was purchased from Santa Cruz (USA). PTX and Englerin A was purchased from Sigma Aldrich (USA).
5
Isolation and Culture of HUVECs
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HUVECs were enzymatically isolated from human umbilical cord veins as described previously (Jaffe et al. 1973) (link). The endothelial cells were cultured in gelatin (0.1%)-coated culture flasks filled with M199 medium (Welgene, Korea) containing 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 3 ng/ml bFGF, 5 units/ml heparin and 250 μg/ml fungizone. The cells were used in passages two to six for each experiment.
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