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2 protocols using reagent of 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt

1

Optimizing Cell Transfection Protocols

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To ensure high-quality and reliable results, the following materials were used in the study. Ultrapure water with a resistivity of 18.2 MΩ cm was obtained from the Mili-Q water purification system (Milford, MA, USA). Acetonitrile, used for HPLC measurements, was of LC-MS grade and purchased from Burdick & Jackson (Muskegon, MI, USA). Formic acid was used as a mobile phase modifier and was of mass-spectrometry grade and obtained from Sigma-Aldrich (Darmstadt, Germany).
EndoFectin™ Max Transfection Reagent (EFM1004) was purchased from GeneCopoeia, located in Rockville, MD, USA. DEAE-Dextran hydrochloride (#D9885) and Ang II (#A9525) were purchased from Sigma-Aldrich in St. Louis, MO, USA. FMK (#Axon 1848) was purchased from Axon Medchem in VA, USA. Finally, the reagent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was bought from Sigma-Aldrich Chemie, located in St. Louis, MO, USA.
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2

Cytotoxicity Evaluation of Breast Cancer Cell Lines

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Six human breast cancer cell lines including MDA-MB-231, MDA-MB-468, MCF-7, BT-474, HCC-1954, and SK-BR-3 were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown in standard medium supplemented with 10% heat-inactivated fetal bovine serum, 100-IU/mL penicillin, and 100-µg/mL streptomycin. Cell lines were maintained at 37℃ in a humidified atmosphere with 5% CO2. MCF-7, MDA-MB-231, and MDA-MB-468 cells were cultured in medium additionally supplemented with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 10-µg/mL insulin. ABT-737, docetaxel, and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) were purchased from Selleck Chemicals (Houston, TX, USA) and solubilized in dimethyl sulfoxide obtained from Thermo Fisher Scientific (Waltham, MA, USA). Reagent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
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