Human umbilical vein endothelial cells (HUVECs) were obtained from the Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai). HUVECs were kept in RPMI-1640 medium from Invitrogen in Gaithersburg, Maryland, which also contained 10% fetal bovine serum, 2 mM l -glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (complete medium). Human intestinal epithelial cells (HIECs) were supplied by Guangzhou Jennio Biotech Co., Ltd. Both HIEC and HCT116 (ATCC #CCL-247) cell lines were cultured in DMEM (Dulbecco’s modified Eagle’s medium, Thermo Fisher Scientific) containing 10% FBS at 37 °C in a humidified environment consisting of 95% air and 5% CO2. All cell lines were passaged for fewer than 6 months after being revived. Artesunate (S2265), 5-fluorouracil (S1209), and insulin (S6955) were supplied by Selleck. H2O2 (10011208) was purchased from Wokai Biotechnology (Shanghai, China). They were reconstituted in dimethyl sulfoxide (DMSO) and kept at 20 °C for each experiment. The ultimate 5-FU concentration employed was 10 mmol/L in all experiments.
Artesunate
Manufactured by Selleck Chemicals
Sourced in United States
Artesunate is a pharmaceutical product used as a laboratory reagent. It is a semi-synthetic derivative of artemisinin, a natural compound extracted from the plant Artemisia annua. Artesunate is commonly used in scientific research and development applications.
Automatically generated - may contain errors
Lab products found in correlation
Sorafenib, by Selleck Chemicals (2 mentions)
Express protein purification system, by Qiagen (2 mentions)
Soluble recombinant murine trail, by R&D Systems (2 mentions)
Erastin, by Selleck Chemicals (2 mentions)
Dihydroartemisinin, by Selleck Chemicals (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Insulin, by Selleck Chemicals (1 mentions)
5 fluorouracil, by Selleck Chemicals (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Verteporfin, by Selleck Chemicals (1 mentions)
Artemisinin, by Merck Group (1 mentions)
Glutamaxtm, by Thermo Fisher Scientific (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
9 protocols using artesunate
1
Culturing HUVECs, HIECs, and HCT116 Cells
2
Recombinant Human TRAIL Production
3
Antifungal Screening of Artemisinin Analogues
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All C. glabrata strains used in this study are listed in Table S1 . The mutant strains were constructed by standard genetic techniques under a C. glabrata ATCC 55 background, and knockout strains were constructed by homologous recombination following a previously described protocol [19 (link)]. Yeast strains were cultivated in YPD medium (1% yeast extract, 1% peptone, 2% glucose) or YPG medium (1% yeast extract, 1% peptone, 2% glycerol); solid media contained 1.5% agar. FDA-approved drugs used in screening antifungal drugs were purchased from Selleck (Houston, TX, USA), this library included artemisinin and its analogues artesunate, dihydroartemisinin, artemether.
4
Investigating Artesunate and Verteporfin Effects on UM Cell Lines
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The human UM cell lines C918 and M619 were obtained from the Type Culture Collection of the Chinese Academy of Medical Sciences. C918 or M619 cells were incubated in DMEM (cat. no. C11995500BT; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (cat. no. P30-3301; PAN-Biotech GmbH) and 1% penicillin-streptomycin (cat. no. 15140-122; Gibco; Thermo Fisher Scientific, Inc.). All cells were maintained in an incubator at 37°C with 5% CO2 in a water-saturated atmosphere. Artesunate (Fig. 1A ) and verteporfin were procured from Selleck Chemicals (cat. nos. S2265 and S1786, respectively) and used to treat the cells for 48 h. Dimethyl sulfoxide (DMSO; cat. no. 276855; Sigma-Aldrich; Merck KGaA) was used as a negative control.
5
Artemisinin Compound Preparation and Use
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Artemisinin was either synthesized and purified as described in Supplementary Information or purchased (Sigma, Saint Louis, Missouri, USA). Artesunate was purchased (Selleckchem, Houston, Texas, USA or TCI, Eschborn, Germany). Artemether was purchased (Selleckchem, Houston, USA). Compounds were dissolved in DMSO and frozen. Details on sample prepration and application of compounds are provided in Supplementary Information.
6
Artesunate Effects on Cellular Oxidative Stress
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The chemicals used and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Sigma-Aldrich (Taufkirchen, Germany) or Merck (Darmstadt, Germany), unless otherwise stated. Artesunate (CAS 88495-63-0) was obtained from Selleck Chemicals GmbH (Planegg, Munich, Germany). Fetal bovine serum (FBS) was acquired from Pan-Biotech (Aidenbach, Germany). Penicillin/Streptomycin was purchased from Biochrom (Berlin, Germany) and GlutamaxTM from Gibco (Darmstadt, Germany). Hemin (CAS 16009-13-5), Deferoxamine mesylate salt (CAS 138-14-7) and DCFH-DA (CAS 4091-99-0) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Zinc protoporphyrin (CAS 15442-64-5) was acquired from Enzo Life Science (Lörrach, Germany). All reagents for flow cytometric analysis were obtained from Sysmex-Partec (Kobe, Japan). DC™ protein assay kit was purchased from Bio-Rad (Feldkirchen, Germany). Clarity™ Western ECL Substrate was obtained from Bio-Rad Laboratories, Inc., (Hercules, CA, USA).
7
Recombinant Human TRAIL Production
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For production of recombinant human TRAIL, a human TRAIL cDNA fragment (amino acids 114–281) obtained by RT-PCR was cloned into a pET-23d plasmid (Novagen, Madison, WI), and His-tagged TRAIL protein was purified using the Qiagen express protein purification system (Qiagen, Valencia, CA). Soluble recombinant murine TRAIL and cell-permeant pan caspase inhibitor Z-VAD-FMK were purchased from R&D systems (Minneapolis, MN). Liproxstatin-1, deferoxamine, and ferrostatin-1 were obtained from Sigma-Aldrich (St. Louis, MO). Artesunate, erastin, and mitomycin C were purchased from Selleckchem (Houston, TX).
8
Antibody-based Detection of Apoptosis
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Antibody against cleaved caspase 3 was obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody against xCT was purchased from Sangon Biotech (Shanghai, USA). Antibody against β-actin and tubulin were purchased from Bioworld Technology (Philadelphia, PA, USA). Anti-rabbit IgG (H+L) affinity purified secondary antibody was obtained from Vector Laboratories (Burlingame, CA, USA). Artesunate, dihydroartemisinin, N-Acetyl-L-cysteine and ferrostatin-1were acquired from Selleck (Houston, TX, USA). RPMI-1640 medium was purchased from HyClone (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Cromwell, CT, USA). 2′,7′-Dichlorofluorescin diacetate was purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Cytostatic Effects of Sorafenib and Artesunate
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The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the 5-ethynyl-2’-deoxyuridine (EDU) immunofluorescence staining assay (Ribobio, Guangzhou, China) were used to test the propagation capacity of HCC cells. Sorafenib (99.15% purity) and artesunate (99.89% purity) were purchased from Selleck Chemical (United States). Briefly, for the MTT assay the cells were cultured at a density of 6,000 cells/well in 96-well plates in triplicate and treated with Sorafenib and/or artesunate for 48 h. For the EDU immunofluorescence staining assay the cells were incubated with Sorafenib for 24 h before permeabilization. EDU staining and fixation were performed in accordance with the manufacturer’s instructions. A concentration of 1 mg/ml of 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, China) was used to stain the cell nuclei for 15 min. Cell numbers were calculated via confocal laser scanning microscopy (Olympus, FV10i, Japan).
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