2 7 dichlorofluorescein dcfh da

1,6-Hexanediamine (92%), N,N-bis(3-aminopropyl)methylamine (98%), chloroform (99%), sodium hydroxide (50 wt% aqueous solution) and benzyltriethylammonium chloride (TEBAC) were purchased from Wanqing Chemical Instrument Co., Ltd (Nanjing, P.R.C.). Paclitaxel, oxaliplatin, cisplatin (DDP), verapamil (VRP) and z-VAD-FMK were obtained from Selleck Chemicals (Houston, USA). Annexin V-FITC/PI double-staining kit was provided by KeyGen (Nanjing, P.R.C.). N-acetyl-L-cysteine (NAC), hemoglobin, Hoechst 33342, crystal violet, 4’,6-diamidino-2-phenylindole (DAPI), 3-amino-4-aminomethyl-2’,7’-difluorescein diacetate (DAF-FM DA) and 2’,7’-dichlorofluorescein (DCFH-DA) were provided by Beyotime Biotechnology (Nanjing, P.R.C.). Propidium iodide (PI), calf thymus DNA (ct-DNA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and ethidium bromide (EB) were purchased from Sigma-Aldrich (St. Louis, USA). γ-H2AX (phospho S139) antibody was obtained from Abcam (Cambridge Science Park, Cambridge, UK). Primary antibodies against specific for cleaved caspase-3, cleaved caspase-9, Bax, poly (ADP-ribose) polymerase (PARP), β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Boston, USA).

PMVEC apoptosis was measured by flow cytometry using an Annexin V and propidium iodide staining kit (BD Pharmingen™, Cat: 556547, USA) according to the manufacturer’s instructions. The level of intracellular ROS in PMVECs was measured by 2′,7′-dichlorofluorescein (DCFH-DA) (Beyotime, S0033, China) following the recommended protocols. Analyses were performed with flow cytometry (BD FACSAria™ III system, USA). Levels of intracellular ROS were quantified by the mean fluorescence intensity (MFI).