2 7 dichlorofluorescein diacetate dcfh da

Intracellular reactive oxygen species (iROS) were determined using the fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Molecular Probes, Eugene, OR, USA), and mitochondrial-specific ROS were measured by MitoSOX Red (Molecular Probes). Briefly, dual-biofilm cells treated with and without 12.5 μg mL⁻¹ of MYR after centrifugation at 13,000× g for 5 min, were treated with 10 mM H₂DCFDA for 1 h, or 5 M MitoSOX Red (Molecular Probes), for 30 min at 37 °C. The fluorescent cells were measured with the FACS Verse microplate reader [15 (link)].

Casticin was purchased from Biopurify Phytochemicals Ltd. (Chengdu, China). The compound has a molecular weight of 374.3, appears as yellow crystals and has a purity of 98.0%. Casticin was prepared in dimethyl sulfoxide (DMSO) as a 10 mmol/l stock solution and diluted in medium to the indicated concentration prior to use. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Eugene, OR, USA). Propidium iodide (PI), ethidium bromide, N-acetylcysteine (NAC, an oxygen-free radical scavenger) and SP600125 (a JNK inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-JNK1 and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-phosphorylated JNK1/2, -phosphorylated ASK1, -ASK1, -phosphorylated Bim-extra long (EL) and -Bim-EL antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

DHA, DAPI, 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) and Oil red O were purchase from Sigma (St. Louis, MO, USA). The 2, 7-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR), and z-VAD-FMK was obtained from Calbiochem (La Jolla, CA, USA). The anti-caspase-8 antibody (9746) and the anti-CHOP/GADD153 (2859) antibodies were obtained from Cell signaling technology (Beverly, MA, USA). The anti-PARP-1 (sc-7150) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-KDEL antibody (ADI-SPA-827) was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The anti-DR5 antibody (AB16942) was purchase from Millipore (Burlington, MA, USA).

Gen and Rev were provided by Xi’an QingYue Biotechnology Co. Ltd. (China) and Sigma Chemical Co. (St. Louis, MO, USA), respectively. As₂O₃ was acquired from Harbin YI-DA Pharmaceutical Limited Company. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), cell-penetrating lipophilic cationic fluorochrome JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine), the Total Superoxide Dismutase Assay Kit with 2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), and Annexin V-FITC Apoptosis Detection Kit were bought from Beyotime Institute of Biotechnology (China) and stored at −20°C in the dark. The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was provided by Molecular Probes (Eugene, OR, USA). The TUNEL detection kit was purchased from Roche (Cell Death Detection Kit; Roche Biochemicals; Mannheim, Germany). LC3A/B monoclonal antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The inert gases helium (He), neon (Ne), argon (Ar), krypton (Kr), and xenon (Xe) were bought from Hokusan Co., Ltd. (Toyama, Japan). All gases were of pure grade (≥ 99.9%). The spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Labotec Co., Ltd., Tokyo, Japan), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M₄PO; Sigma Aldrich Chem. Co., MO), phenyl N-t-butylnitrone (PBN; Sigma Aldrich Chem.), 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS; Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; Dojindo, Kumamoto, Japan) were used to detect the presence of different free radical species in ultrapure water following exposure to CAP by EPR spin trapping.
Thymine, thymidine, uracil, uridine, sodium acetate, and L-alanine were obtained from Wako Pure Chemical Industries, Ltd. Hydroxyphenyl fluorescein (HPF), aminophenyl fluorescein (APF), and diaminofluorescein-2 (DAF-2DA) were from Sekisui Medical Co., Ltd. (Tokyo, Japan). Hydroethidine (HE) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were from Molecular Probes Inc. (Eugene, OR).

EA.hy926 (EAHY) endothelial cells were kindly provided by Professor Cora-Jean S. Edgell (Pathology Department, University of North Carolina, USA) and were previously described by Tseng et al. [40] . Human U937 mononuclear cells were obtained from American Type Culture Collection [41] . EAHY and U937 cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) and RPMI1640, respectively supplemented with 10% fetal bovine serum (FBS), antibiotics and glutamine (Sigma Chemical Company, USA). ELISA kits for PAI-1, uPA and suPAR were purchased from R&D Systems (Minneapolis, MN, USA). PGF_2α ELISA kits were obtained from Cayman Chemical (Ann Arbor, MI, USA). P-cresol and 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Invitrogen Detection Technologies, Grand Island, NY, USA). Flow cytometry reagents were from Becton Dickinson (Franklin Lakes, New Jersey, USA).

About 2 × 10⁵ cells/ml of HepG2 or fibroblast cells were grown in RPMI medium and seeded into a rounded bottom 96-well plate for 24 h. Following incubation, cells were tested in the presence (15.6 μM) or absence of the selected bacteriocin for 24 h. The intracellular induced ROS was assessed using the fluorescent membrane permeable probe 2,7-dichlorofluorescein diacetate (DCFH-DA) (Molecular Probes, Sigma Aldrich). The fluorescent probe was added at the end of the treatment in a final concentration of 20 μM. After incubation, the resulted fluorescence was measured using flow cytometry (Sawada et al., 1998 (link); El-Adawi et al., 2012 (link)).