A lactate dehydrogenase (LDH) release assay was performed using an LDH Cytotoxicity Detection Kit (Takara Bio, Shiga, Japan). Doxorubicin (Wako) at 0.1, 1, 2, 5, or 10 μM was added for 24 h. After adding Doxorubicin for 24 h, the supernatants were then collected, and a mixture of diaphorase/NAD+ was added to each well. After incubation in the dark room for 30 min at room temperature, the absorbance was recorded on a microplate reader (DS Pharma Biomedical, Osaka, Japan) at a wavelength of 490 nm with a reference wavelength of 600 nm. The experiments were performed in triplicate. The LDH release was determined as the percentage of LDH release compared with that of the vehicle control.
Doxorubicin
Manufactured by Fujifilm
Sourced in Japan, United States
Doxorubicin is a type of laboratory equipment used for scientific research and analysis. It is a chemical compound commonly used in medical and biological applications.
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Lab products found in correlation
Paclitaxel, by Fujifilm (6 mentions)
Cisplatin, by Fujifilm (4 mentions)
Carboplatin, by Fujifilm (2 mentions)
Docetaxel, by Fujifilm (2 mentions)
Thapsigargin, by Fujifilm (2 mentions)
Etoposide, by Fujifilm (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Vinblastine, by Fujifilm (1 mentions)
Rhodamine 123, by Fujifilm (1 mentions)
Tariquidar, by Adooq (1 mentions)
125i iodoarylazidoprazosin iaap, by PerkinElmer (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Calcein am, by Merck Group (1 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
Recombinant egf, by Merck Group (1 mentions)
Cyp1a2, by Merck Group (1 mentions)
Anti flag antibody conjugated agarose, by Merck Group (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Erlotinib, by Cayman Chemical (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
26 protocols using doxorubicin
1
Doxorubicin-Induced Cytotoxicity Assay
2
Cytotoxic Drug Preparation Protocol
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Methyl methanesulfonate (MMS) and thiabendazole (TBZ) were purchased from Sigma-Aldrich (St Louis, MO). Camptothecin (CPT), hydroxyurea (HU), doxorubicin and cisplatin were from Wako Pure Chemical Industries Ltd (Osaka, Japan). Vorinostat/suberoylanilide hydroxamic acid (SAHA) was synthesized in-house (see Supplementary Materials )69. doxorubicin was dissolved in water, SAHA (refer supplementary procedure ) in DMSO, and cisplatin (Wako, Pure Chemical Industries, Ltd, Osaka, Japan) in 10% NaCl. All drugs were further diluted with their respective solvents prior to use according to manufacturers’ recommendations.
3
Irradiation and Doxorubicin Effects on CHMp and CHMm Cell Lines
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Irradiation of CHMp and CHMm cell lines was carried out using a Faxitron CP160 irradiator (Faxitron X-ray Corporation, Tucson, AZ, USA) at 5 Gy/min, for a total maximum dose of 15 Gy. Doxorubicin stimulation was applied to the CHMp and CHMm cell lines at suitable concentrations of Doxorubicin (FUJIFILM Wako Pure Chemical Corporation) in the medium.
4
Evaluating Drug Transport Inhibitors
5
EGF, CYP, and Drug Binding Assay
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Recombinant EGF, CYP1A2 and CYP3A4 proteins were purchased from Sigma. Erlotinib was purchased from Cayman. Doxorubicin was purchased from Wako. Anti-FLAG (M2) antibody, FLAG peptide and anti-FLAG antibody-conjugated agarose were purchased from Sigma. Haemin and protoporphyrin-IX (PP-IX) were purchased from Porphyrin Science.
6
Anticancer Compound Stock Preparation
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Hydroxyurea was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and dissolved in distilled water to prepare a 1 M stock solution. Gemcitabine, irinotecan, carboplatin and doxorubicin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and dissolved in dimethylsulfoxide (DMSO) to prepare 1 mM, 20 mM, 25 mM and 10 mM stock solutions, respectively. Methotrexate was also purchased from Wako and dissolved in 1 M NaOH to prepare a 10 mM stock solution. Sunitinib, 5-fluorouracil, paclitaxel and cisplatin were purchased from Sigma (St. Louis, MO, USA) and dissolved in DMSO to prepare 10 mM, 10 mM, 1 mM and 100 mM stock solutions, respectively. Temozolomide was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA) and dissolved in DMSO to prepare a 50 mM stock solution. Antibodies such as Cleaved Caspase-3 (Asp175, #9661), Cleaved PARP (Asp214, #9541), Merlin (#12888), Vimentin (#5741), phospho-Histone H3 (S10, #9706), Cleaved PARP (Asp214, Fluorescein conjugate, #9547), and GAPDH (#5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
7
TNBC Cell Line Manipulation and Analyses
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Triple-negative breast cancer cells, BT549 wild type (WT) cells were purchased from the ATCC by the Laboratory of Experimental Pathology, University of Tsukuba. BT549 TMEPAI-knocked-out (KO) cells were established previously using the CRISPR-Cas9 technique.
Cells were cultured and maintained using culture medium described previously.16 (link) Experiments were done when the cells reached 70% confluency under cell starvation in 1% Fetal Bovine Serum (Gibco) in a 10-cm tissue-culture dish (Corning). Cells were then treated with recombinant human TGF-β (Wako) 2 ng/mL for 24 hours, followed by TGF-β 2 ng/mL and doxorubicin (Wako) 12.9 nM for another 24 hours. The dose of doxorubicin was based on our previous experiment that showed the IC50 of doxorubicin in BT549 cells was 12.9 nM.16 (link) Afterward, cells were harvested and counted. Cell viability was calculated using the trypan blue exclusion method. Then, these cells were lysed and isolated for RNA and protein for further analysis. All the treatments were done in four separate experiments in duplicate.
Cells were cultured and maintained using culture medium described previously.16 (link) Experiments were done when the cells reached 70% confluency under cell starvation in 1% Fetal Bovine Serum (Gibco) in a 10-cm tissue-culture dish (Corning). Cells were then treated with recombinant human TGF-β (Wako) 2 ng/mL for 24 hours, followed by TGF-β 2 ng/mL and doxorubicin (Wako) 12.9 nM for another 24 hours. The dose of doxorubicin was based on our previous experiment that showed the IC50 of doxorubicin in BT549 cells was 12.9 nM.16 (link) Afterward, cells were harvested and counted. Cell viability was calculated using the trypan blue exclusion method. Then, these cells were lysed and isolated for RNA and protein for further analysis. All the treatments were done in four separate experiments in duplicate.
8
Characterization of Fission Yeast Ess1 Mutant
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Schizosaccharomyces pombe SPBC2D10.03c/Ess1 was identified to be the counterpart of human C1orf123 by Pombase and NCBI BLASTp. The null mutant was obtained from Bioneer (ver 2.0) haploid gene deletion library (Bionee, Daejeon, South Korea) and a strain without nutritional markers was created by crossing with prototrophic wild-type (WT) 972 strain. PCR with locus specific primers were performed to confirm the gene deletion. Previously published procedures to culture and test drug hypersensitivity in fission yeast were followed (Tay et al., 2013 (link); Nguyen et al., 2016 (link)). Briefly, cells were grown in YEA (3% glucose, 0.5% yeast extract, 75 mg/ml L-adenine) to log-phase, ten-fold serial-diluted and spotted onto media agar plates incorporated with drugs: hydrogen peroxide (H2O2), hydroxyurea (HU) (Sigma-Aldrich) and doxorubicin (Wako Pure Chemical Industries Ltd, Japan). Cell growth was documented 3 and 6 days after spotting.
9
Tumor Sphere Drug Sensitivity Assay
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One hundred CR cells in 100 µl of complete F-medium were seeded in ultra-low attachment 96-well plates (Corning) to generate tumorspheres. The tumorspheres were treated with DMSO, 4-OH-tamoxifen (Sigma-Aldrich), doxorubicin (WAKO), docetaxel (WAKO), paclitaxel (WAKO), bevacizumab (Selleck), fulvestrant (Selleck) or palbociclib (Selleck) and incubated at 37 °C in 5% CO2. After 48 h of treatment, the CellTiter-Glo® 3D cell viability assay (Promega, WI, USA) was used to assess growth inhibition. To determine the IC50, cells were treated for 4 days with 4-OH-tamoxifen. was also used for drug sensitivity assay.
10
Migracin A inhibits VEGF and IGF-1 signaling
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Migracin A was isolated from Streptomyces sp. as reported previously [1 (link)]. Doxorubicin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Recombinant Human VEGF165 was purchased from R&D Systems (Minneapolis, MN). IGF-1 receptor kinase inhibitor Linsitinib and PI3K/Akt inhibitor LY294002 were purchased from Chemie Tek (Indianapolis, IN) and Wako Pure Chemical Industries, respectively.
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