The cytotoxic effect of BV (melittin comprise approximately 50% and apamin comprise 3% of dried BV) (Chung Jin Biotech Co., Ansan, Korea) [26 (link)], melittin (Enzo Life Sciences AG, Lausen, Switzerland), and apamin (Sigma-Aldrich, St. Louis, Mo, USA) was evaluated using a CellTiter-96® aqueous cell proliferation assay kit (Promega, Madison, WI, USA). On a 96-well microstate plate, NP fibroblasts were cultured in the presence of 0.1, 1, 3, and 5 μg/mL of BV, 0.1, 1, 3, and 5 μg/mL of melittin, and 0.1, 1, 5, and 10 μg/mL of apamin for 24 h at 37 °C in a 5% CO2. The reduced tetrazolium compound produces a colored formazan product due to the mitochondrial activity in the cell. The amount of formazan is directly proportional to the number of viable cells. Color intensities were assessed with a fluorescence microplate reader at 490 nm.
Melittin
Manufactured by Enzo Life Sciences
Sourced in United States
Melittin is a naturally occurring peptide derived from bee venom. It functions as a pore-forming agent, capable of disrupting cell membranes.
Automatically generated - may contain errors
Lab products found in correlation
Polymyxin b, by Merck Group (2 mentions)
Gentamicin, by AppliChem (2 mentions)
Tetracycline, by AppliChem (2 mentions)
Celltiter 96 aqueous cell proliferation assay kit, by Promega (1 mentions)
Apamin, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Hacat cell line, by Cytion (1 mentions)
Bee venom, by Merck Group (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Penicillin, by Cytion (1 mentions)
Recombinant human tnf α, by R&D Systems (1 mentions)
Pg lps, by InvivoGen (1 mentions)
Meropenem, by Merck Group (1 mentions)
Aztreonam, by Merck Group (1 mentions)
Rifampicin, by Merck Group (1 mentions)
Ciprofloxacin, by AppliChem (1 mentions)
8 protocols using melittin
1
Cytotoxic Effect of Bee Venom Components
2
Melittin from Bee Venom Bioactivity
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BV was supplied by Chung Jin Biotech Co. (Ansan, Korea) and melittin was supplied by Enzo Life Sciences (Farmingdale, NY, USA).
3
Bee Venom and Melittin Extraction
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Bee venom was supplied by Chung Jin Biotech Co. (Ansan, Korea) and melittin by Enzo Life Sciences (Farmingdale, NY, USA)
4
Bee Venom and Melittin Effects on HaCaT Cells
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The human keratinocyte HaCaT cell line (Cell Lines Service GmbH, Eppelheim, Germany) was cultured in high glucose DMEM supplemented with 10% (v/v) FBS and antibiotics (100 U·mL−1 penicillin and 100 μg·mL−1 streptomycin) at 37°C in a humidified 5% CO2 incubator. HaCaT (5.0 × 105 cells·mL−1) cells were seeded in the complete medium. Twenty‐four hours later, the medium was changed to serum‐free medium with bee venom (1, 10 and 100 ng·mL−1; Sigma) and melittin (0.1, 0.5 and 1 μg·mL−1; Enzo, Plymouth, PA, USA). After 30 min, the cells were stimulated with 10 ng·mL−1 TNF‐α/IFN‐γ for 9 h. PBS (pH 7.4), FBS, DMEM, penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA). Recombinant human TNF‐α and IFN‐γ were purchased from R&D Systems (Minneapolis, MN, USA).
5
Melittin and LPS modulate HaCaT cells
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HaCaT cells (CLS, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GE Healthcare Life Sciences HyClone Laboratories, South Logan, UT, USA) supplemented with 10% fetal bovine serum and 1% antibiotics at 37 °C in a humidified 5% CO2 incubator. The HaCaT cells were seeded at 1.0 × 106 cells per 3 mL of complete medium in a 100-mm TC-treated cell-culture dish. The medium was changed 24 h later with serum-free medium containing the indicated concentrations of melittin (0.1, 0.5, and 1 μg/mL; Enzo Life Sciences, Farmingdale, NY, USA). After 1 h, the cells were co-treated with 100 ng/mL of PgLPS (InvivoGen, San Diego, CA, USA). After 7 h, the cells were collected for the next experiment.
6
Retro-inverse TAT-RasGAP peptide characterization
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The retro-inverse TAT-RasGAP317-326 peptide (amino acid sequence DTRLNTVWMWGGRRRQRRKKRG) and its N-terminal FITC-labeled derivative were provided by SBS Genetech (Beijing, China) and stored at −20°C. Melittin was provided by Enzo Life Science (Farmingdale, NY), polymyxin B, meropenem and aztreonam by Sigma-Aldrich (Burlington, MA), and gentamicin and tetracycline by Applichem (Darmstadt, Germany).
7
Melittin Modulation of Cytokine-Induced HaCaT Response
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HaCaT (CLS, Eppelheim, Germany) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics at 37 °C in a humidified 5% CO2 incubator. The HaCaT cells were seeded at 1.0 × 106 cells per 3 ml complete medium in a 100 mm TC-treated cell culture dish. The medium was changed 24 h later with a serum-free medium containing the indicated concentrations of melittin (0.1, 0.5, and 1 μg ml−1; Enzo Life Sciences, Farmingdale, NY, USA). After 1 h, the cells were co-treated with 50 ng/mL each of human recombinant IL-4 and IL-13 (R&D systems, Minneapolis, MN, USA). After 23 h, the cells were collected for the next experiments.
8
Antimicrobial Retro-Inverso Peptide TAT-RasGAP
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TAT-RasGAP317-326 is a retro-inverso peptide (i.e. reversed direction compared to natural sequence including D-amino acids) with an antimicrobial activity [20] composed of amino acids 48-57 of HIV TAT protein (RRRQRRKKRG) and 317-326 of human RasGAP protein (DTRLNTVWMW) linked with two glycines. TAT-RasGAP317-326 was synthesized by SBS Genetech (Beijing, China). Ciprofloxacin, tetracycline and gentamicin were from Applichem (Darmstadt, Germany), rifampicin and polymyxin B from Sigma-Aldrich (Saint-Louis, MO), and melittin from Enzo Life Sciences (Farmingdale, NY).
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