MDA-MB-231 (ATCC HTB-26), MDA-MB-468 (ATCC HTB-132) and SK-BR-3 (ATCC HTB-30) were cultured in DMEM/F12 (Sigma Aldrich) with 10% fetal bovine serum (FBS) (Life technologies); MCF-7 (ATCC HTB-22) in DMEM with 10% FBS; and BT-474 (ATCC HTB-20) in DMEM/F12 with 10% FBS and insulin (0.01 μg/ml). Cell lines were grown as monolayers at 37°C in a 5% CO2 atmosphere. Media were supplemented with L-glutamine (2 nM) (Gibco), penicillin G (100 U/ml), and streptomycin (100 μg/ml) (Gibco). Cells were purchased from American Type Culture Collection (ATCC) and authenticated (LGC Standards). Reagents: doxorubicin (10 μM) (Sigma Aldrich), okadaic acid (OA) (2.5 nM) and FTY720 (10 μM) (Calbiochem). To generate doxorubicin-resistant cells, MDA-MB-231 cells were cultured in the presence of increasing doses of doxorubicin (three subculturing cycles per concentration), starting at 0.5 μM. In order to assess the evolution of resistance, we determined the IC50 after every doxorubicin concentration point, by using an MTS assay (Promega) at 24 h of treatment. The resistance of every doxorubicin-resistant clone was defined as the ratio between resistant and parental cells IC50 values.
Doxorubicin
Manufactured by Promega
Doxorubicin is an anthracycline antibiotic used in laboratory research. It is a cytotoxic agent that intercalates with DNA, inhibiting DNA and RNA synthesis. Doxorubicin is commonly used in cell and molecular biology experiments to study the effects of DNA damage and cell death.
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Lab products found in correlation
Doxorubicin, by Merck Group (5 mentions)
Epirubicin, by Merck Group (2 mentions)
Docetaxel, by Merck Group (2 mentions)
Epirubicin, by Corning (2 mentions)
Paclitaxel, by Promega (2 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay solution, by Promega (2 mentions)
Safire2 plate reader, by Tecan (2 mentions)
96 well plate, by Corning (2 mentions)
Paclitaxel, by Merck Group (2 mentions)
Docetaxel, by Promega (2 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Dmem f12, by American Type Culture Collection (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Mts assay, by Promega (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Bt549, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
6 protocols using doxorubicin
1
Generating Doxorubicin-Resistant Breast Cancer Cells
2
Cytotoxicity of Chemotherapeutics in TNBC
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Human triple negative breast cancer MDA-MB231 and BT549 cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). MDA-MB231 cells were cultured in L15 supplemented with 10% FBS (Mediatech), and BT549 cells were maintained in RPMI medium 1640 supplemented with 10% FBS (Mediatech). Epirubicin, doxorubicin, paclitaxel and docetaxel were purchased from Sigma-Aldrich (Milwaukee, WI). Drugs were dissolved in DMSO and aliquots of stock solutions were frozen at −80 °C. Cell proliferation assays were performed in triplicate at each drug concentration. Cytotoxicity assays with the lymphoblastoid and tumor cell lines were performed in triplicate at each dose. Specifically, 90 μL of cells (5 × 103 cells/mL) were plated into 96-well plates (Corning, NY) and were treated with 10 μl of Epirubicin or doxorubicin at final concentrations of 0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.55, 1, and 2 μM. Similarly, cells were treated with paclitaxel or docetaxel at 0, 0.01, 0.1, 1, 10, 50, 100, 1000, and 5000 nM. After incubation for 72 hours, 20 μL of CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay solution (Promega Corporation, Madison, WI) was added to each well. Plates were read in a Safire2 plate reader (Tecan AG, Switzerland).
3
Studying OCT-1 Modulation in Cancer Cells
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MDA-MB231 cells were cultured in the complete DMEM medium. ER stress was induced by culturing cells in the presence of 1 µg/mL of tunicamycin for 24 h. To study the role of total OCT-1 or OCT-1A isoform knockdown in cell response to hypoxia or doxorubicin (Sigma-Aldrich) and docetaxel (Sandoz), cells were placed into the 96-well plates containing the complete DMEM medium in the number of 30,000 cells per well in five replicates. Then, 24 h later, cells were cultured in the complete DMEM medium at 370 °C with 0.5% O2 (hypoxia) for 24 h or in the presence of 1 µg/mL of doxorubicin for 48 h. Cell viability under hypoxia or in the presence of doxorubicin was measured using the CytoTox-Glo Cytotoxicity Assay (Promega). The percentage of live cells relative to the total number of cells and mean values were calculated.
4
Cytotoxicity of Doxorubicin on PANC1 Cells
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A non-radioactive cell proliferation assay kit® (Promega Corporation) was used to assess the cytotoxicity of doxorubicin (Ebewe) on PANC1 cells by measuring the cell titer. An MTT proliferation assay was performed for the control PANC1 cells and for the cells exposed to the hypoxic conditions after 10 and 20 cycles of acute hypoxia, and after 5 cycles of chronic hypoxia.
The cytotoxicity of doxorubicin was determined using an MTT assay. Briefly, cells were seeded at an initial density of 7-10x103 cells/well in 96-well culture plates (Costar) in 100 µl complete culture medium and incubated in a humidified incubator supplied with 5% CO2 at 37˚C for 24 h. Cells were incubated in a stock solution of doxorubicin and dilutions thereof (8x10-10 to 1x10-4 M) were prepared in DMEM high glucose medium in a humidified incubator supplied with 5% CO2 at 37˚C for 72 h. The solutions were then removed and replaced with 100 µl fresh DMEM to which 100 µl MTT solution was added to each well, followed by incubation at 37˚C for 3 h. MTT-media solution was then removed and 100 µl MTT stop solution was added to dissolve the dark blue formazan crystals. Absorbance was measured at 570 nm using a microplate reader (Synergy™ HTX, BioTek Instruments Inc.), and the IC50 values of doxorubicin when used to treat cells were calculated.
The cytotoxicity of doxorubicin was determined using an MTT assay. Briefly, cells were seeded at an initial density of 7-10x103 cells/well in 96-well culture plates (Costar) in 100 µl complete culture medium and incubated in a humidified incubator supplied with 5% CO2 at 37˚C for 24 h. Cells were incubated in a stock solution of doxorubicin and dilutions thereof (8x10-10 to 1x10-4 M) were prepared in DMEM high glucose medium in a humidified incubator supplied with 5% CO2 at 37˚C for 72 h. The solutions were then removed and replaced with 100 µl fresh DMEM to which 100 µl MTT solution was added to each well, followed by incubation at 37˚C for 3 h. MTT-media solution was then removed and 100 µl MTT stop solution was added to dissolve the dark blue formazan crystals. Absorbance was measured at 570 nm using a microplate reader (Synergy™ HTX, BioTek Instruments Inc.), and the IC50 values of doxorubicin when used to treat cells were calculated.
5
Assessing Cell Viability and Apoptosis
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3T3 MEFs, 293Ts and HeLa cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 25mM Glucose and 4mM L-Glutamine supplemented with 10% (v/v) fetal bovine serum (FBS) (FBS, Gemini BioProducts), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. For doxorubicin (Sigma), and CPT (Sigma) treatment, drugs were added to the medium at the indicated doses. At the desired time points, cells were collected by trypsinization and incubated with propidium iodide (PI, 1 μg/mL; Molecular Probes). Cell death was determined using flow cytometry by PI exclusion. To examine recovery following exposure to drugs, cells were treated with CPT for 1.5 h, then washed and fed with fresh medium with no drugs, and cultured for the indicated periods of time. For the colorimetric MTS-PMS assay (Promega), cells were seeded at 10,000 cells/well in a 96-well plate followed by doxorubicin treatment at the indicated concentrations for 24 hours. 20 μl of MTS/PMS solution (final concentrations 333 μg/ml MTS and 25 μM PMS) were added to 100 μl of media per well. Cells were incubated with the MTS-PMS reagent for 1hr at 37°C. Absorbance was measured at 490nm with a spectrophotometer.
6
Cytotoxicity Screening of Chemotherapeutics
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