4 amino 5 methylamino 2 7 difluorofluorescein diacetate daf fm da

Cellular superoxide generation was detected using the fluorescent probe 2′,7′-Dichlorofluorescin diacetate (DCF-DA; Sigma-Aldrich, St. Louis, MO, USA) as previously described [13 (link)]. Treated HUVECs were treated with DCF-DA (10 μmol/L) for 30 min at cell culture temperature, then imaged using fluorescence microscopy. Next, to quantify reactive oxygen species (ROS) levels, HUVECs were seeded in 96 wells and treated as described, following which the fluorescence level was measured using fluorescence intensity of Ex/Em = 485/535 nm in a fluorescence reader (Versamax, Molecular Devices, Sunnyvale, CA, USA). Subsequently, NO production was measured using 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM DA; Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 5 μM. After the washing process, the degree of NO production was quantified by completing the intracellular de-esterification reaction and measuring the fluorescence at Ex/Em 495/515 nm.

The production of reactive oxidative species (ROS) and reactive nitrogen species (RNS) was respectively examined using the 2′,7′-Dichlorofluorescein Diacetate (DCFH-DA) (Invitrogen, California, USA) and 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FMDA) (Thermo Fisher, Carolina, USA) [36 (link)].

Abaxial leaf epidermal peels from 4-week-old plants were used to determine H₂O₂ and NO production. Epidermis was incubated in the stomatal opening solution containing ABA or BL alone, or in combination, and were then incubated in 100 µM 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) (Fisher Scientific) for 15 min to detect H₂O₂ production. To detect NO production, epidermis was incubated with 200 µM 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FMDA) (Thermo Fisher Scientific) for 20 min. Dyes were washed off with distilled water. H₂O₂ and NO production in guard cells was observed under a fluorescence microscope (Leica, DM2500 with a fluorescence module; Fluo Illuminator L4/23) and the L5 filter system (excitation BP480/40, emission BP527/30). To prevent photo-oxidation of H₂DCF-DA, all fluorescence images were collected with a single rapid capture (150.8 ms frame^–1) at ×400 magnification, as described by Shang et al. (2016) (link).

Whole blood was incubated in the absence or presence of olaparib (0.1, 1, and 10 μM) for 4 h. ROS and NO generation were measured constitutively and after stimulation with heat-killed S. aureus for 30 min. ROS and NO levels in monocytes and neutrophils were quantified by measuring the oxidation of 2,7-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich) and 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FMDA; Invitrogen, Carlsbad, CA, USA), respectively, using flow cytometry. Briefly, the tubes from each sample were incubated in the presence of 0.06 mM DCFH-DA or 0.01 mM DAF-FMDA in a 37 °C shaking water bath for 30 min. After incubation, 2 mL of 3 mM EDTA (Sigma-Aldrich) or PBS was added to each tube for ROS and NO determination, respectively, and the mixture was centrifuged at 800× g for 5 min at 4 °C. Erythrocytes were lysed in hypotonic saline, and the pellets were incubated with 5 μL of CD14 antibody (BV-711; BD Bioscience) at room temperature for 15 min in the dark. Two milliliters PBS was added to each tube, and the mixture was centrifuged at 800× g for 5 min at 4 °C. The supernatants were discarded, and the pellets were resuspended in 300 μL PBS for flow cytometry analysis.

L-arginine, tetrahydrobiopterin (BH₄), sulfanilamide (SA), N-naphthyl-ethylenediamine (NED), c-Myc agarose column and c-Myc antibody were all purchased from Sigma-Aldrich. The NOS inhibitor L-NAME was obtained from Calbiochem (San Diego, CA). The polyclonal anti-SyNOS was acquired from Genscript (USA). Restriction enzymes, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA), Bacto-yeast extract, IPTG and Escherichia coli DH5α cells were purchased from Invitrogen (USA). BL21 (DE3) pLys cells, and both pET24b and pET15b were obtained from Novagen (Madison, WI). Synechococcus PCC 7335 was purchased as part of the Pasteur Culture Collection (PCC, Paris, France).