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

1

In Vitro Cell Cytotoxicity Evaluation

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Dulbecco’s modified Eagle’s medium (DMEM) without phenol red, fetal bovine serum (FBS), penicillin/streptomycin, and trypsin (2.5%) were obtained from Gibco® (Thermo Fisher Scientific, Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was procured from Bio Basic (Amherst, NY, USA). The antioxidant standards for the HPLC analysis together with other chemicals and buffers, such as DMSO, dichloro-dihydro-fluorescein diacetate (DCFH-DA), dihydroethidium (DHE), and Purpald®, were obtained from Sigma-Aldrich (Merck, Darmstadt, Germany).
The anti-mouse IgG conjugated with HRP was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). All the antibodies for the Western blot experiments were obtained from Cell Signaling Technology® (Danvers, MA, USA.). All the other reagents and chemicals were purchased from Merck® (Darmstadt, Germany). The cell culture wares, including plates and flasks (polystyrene coated), were obtained from Nunc Cell Culture® (Thermo Fisher Scientific, Waltham, MA, USA).
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2

Molecular Mechanisms of Oxidative Stress

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2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) was purchased from Sigma‐Aldrich (St Louis, MO, USA). Hoechst 33342 reagent, Dulbecco's modified Eagle's medium (DMEM), and FBS were obtained from Invitrogen (Carlsbad, CA, USA). Primary antibodies for total‐JNKs and phosphor‐JNKs, total‐ERK1/2 and phosphor‐ERK1/2, total‐p38 MAPK and phosphor‐p38 MAPK, total‐Akt and phosphor‐Akt, total‐c‐Jun and phosphor‐c‐Jun, B‐cell lymphoma‐2 (Bcl‐2), Bcl‐2‐associated X (Bax), cleaved caspase‐3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Primary antibody against GAPDH was from Bioworld Technology (Bloomington, MN, USA). 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) reagent was from Bio Basic Inc (Markham, ON, Canada). Anisomycin (Aniso) was from MedChemExpress (New Jersey, MA, USA). Protease inhibitor cocktail was from Roche (Mannheim, Germany). BCA protein assay kit and SuperSignal West Pico chemiluminescent substrate were obtained from Pierce (Rockford, IL, USA). Lactate dehydrogenase (LDH) detection kit was from Jiancheng Bioengineering (Nanjing, China). TUNEL assay kit was from Beyotime Biotechnology (Nantong, China).
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