Daf fm da

The production of NO and ROS in zebrafish larvae was visualized using 4-amino-5-methylamino-2'7'-difluorofluorescein diacetate (DAF-FM-DA, Sigma-Aldrich Chemical Co.) and DCF-DA, respectively, 24 h after chemical treatment as previously described (Jeong et al., 2018[19 (link)]). In brief, zebrafish embryos (4 dpf) were transferred to 24-well plates and incubated with 5 µM DAF-FM-DA and 20 µM DCF-DA for 30 min and visualized using the CELENA^® S Digital Imaging System (Logos Biosystems, Anyang, Gyeonggido, Republic of Korea). Fluorescence intensities were calculated using ImageJ software (Wayne Rasband, National Institute of Health, Bethesda, MD, USA) and expressed as a percentage compared to the untreated control.

Freshly prepared protein extracts prepared as for western blotting were used to assay NO content. An amount of 20 μL of each protein extract was incubated with 180 μL of a solution containing 10 μM of DAF-FM DA (Sigma-Aldrich) in 50 mM HEPES buffer pH 7.5 in microtiter plates, following the method described in [49 (link)]. Samples were incubated at 37 °C for 2 h in the dark. After incubation, the emitted fluorescence of each well was measured in a Varioskan LUX Multimode Microplate Reader (ThermoFisher Scientific, Waltham, MA, USA). Samples were normalized by their total protein content (Table S3) and against a control condition in each experiment. Blanks included in all experiments behaved similarly and emitted a negligible signal, that was subtracted from all experimental samples.
Detection of NO through confocal microscopy was performed using the same experimental conditions and DAF-FM DA staining protocol for the former spectrofluorometry measurements. λ scan 500–666 nm was used to set the emission window and FIRE LUT to represent a fluorescence heatmap of intensity.

Levels of intracellular ROS and NO and extent of lipid peroxidation in primary murine keratinocytes were determined using H₂DCF-DA (Invitrogen/Life Technologies), DAF-FM-DA (Sigma), or C11-BODIPY^581/591 assays (Invitrogen/Life Technologies), respectively. H₂DCF-DA allows detection of intracellular H₂O₂, but it also detects oxygen radicals [52 (link)]. DAF-FM-DA is a probe to detect NO, but it only works under aerobic conditions and it is likely to react with an oxidative product of NO, rather than with NO itself [53 (link)]. Cells were incubated for 30 min with 50 μM H₂DCF-DA or 5 μM DAF-FM-DA, or for 2 h with 2 μM C11-BODIPY^581/591 in cell culture medium at 37°C prior to detachment by trypsin. Fluorescence was directly measured by flow cytometry using the BD Accuri C6 (BD Biosciences, San Jose, CA).

Mpx activity and neutrophils were detected in tg(mpx:GFP)/mpx+/- and tg(mpx:GFP)/mpx-/- larvae at 1 day post E. coli injection (dpi) using Sudan black staining and anti-GFP antibody (molecular probe A11122, dilution 1/500), respectively [21 (link)]. For superoxide detection within the cells, DHE was added to the fish medium at 3 μM at 1 dpi for one hour and larvae were washed 2 times before imaging using confocal microscopy (excitation/emission 532/605 nm) [32 (link)]. To detect nitric oxide, infected tg(lyz:DsRed) embryos were stained with 4-Amino-5-methylamino-2’,7’-difluorofluorescein diacetate, Diaminofluorescein-FM diacetate (DAF-FM-DA) (Sigma, CAS 254109-22-3) [31 (link)] at 5 μM in fish medium for 2 hours at 6, 10 hpi and 1 dpi (for E. coli infection) or 2 hpi (for Salmonella infection). Larvae were rinsed three times in fish water before imaging using epi-fluorescence and confocal microscopy (excitation/emission: 488/515 nm). Dead cells were detected using Sytox Green staining. Larvae were injected with 3 nL of 50 μM Sytox Green (Molecular Probes) in the vein at 1 dpi and placed at 28.5°C. One hour after Sytox Green injection, larvae were mounted in 1% low-melting-point agarose and imaged using epi-fluorescence and spinning disk confocal microscopy (excitation/emission: 488/526 nm).

NO was visualized using the NO-specific fluorescent probe DAF-FM DA (Sigma-Aldrich), according to Wang’s method [70 (link)] with some modifications. Seven-day-old wild-type and mutant seedlings were incubated in 1 ml of 0.5× liquid MS medium (pH 5.8) with 10 μM DAF-FM DA for 20 min. Thereafter, the roots were washed three times for 15 min each in 0.5× liquid MS medium prior to visualization using a fluorescence microscope (ELLIPE TE2000-U; Nikon, Tokyo, Japan). The signal intensities were quantified using MetaMorph (Molecular Devices, Sunnyvale, CA).

The production of NO in HUVECs was measured using diaminofluorescein-FM diacetate (DAF-FM/DA, Sigma-Aldrich) assay. Briefly, the cells seeded in a 96-well black plate at a density of 2 × 10⁴ cells/well were pretreated with DHP1501 (2, 20, or 200 μg/mL) for 90 min before incubation in 5 μM DAF-FM/DA for 1 h at 37°C. 0.5% DMSO was used as a vehicle control, with the relative levels of intracellular NO determined from the fluorescence intensity of DAF-FM at excitation of 486 nm and emission of 520 nm using fluorescence microplate reader (Spark 10M, TECAN).