For fluorescence microscopy, NO and H2O2 were visualized using Diaminofluorescein-FM diacetate (DAF-FM-DA) and 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) probes, respectively (Sigma-Aldrich). Briefly, leaf dices were first loaded with 15 μM DAF-FM-DA for 30 min or 50 μM H2DCF-DA for 10 min in Tris/KCl loading buffer (pH 7.2). The process was performed in darkness at 25 °C. Further, leaves were washed three times (5 min each) using Tris/KCl loading buffer (10 mM Tris and 50 mM KCl, pH 7.2), and visualized under a TE2000-U fluorescence microscope (490 nm excitation; 515 nm emission) (Nikon, Tokyo, Japan). Treatments were repeated at least five times. NO and H2O2 contents were also detected using a Griess reagent (Sigma-Aldrich) and an Amplex red hydrogen peroxide/peroxidase assay kit (Invitrogen), respectively [33 (link)].
Diaminofluorescein fm diacetate daf fm da
Manufactured by Merck Group
Sourced in United States
Diaminofluorescein-FM diacetate (DAF-FM-DA) is a fluorescent indicator used for the detection and measurement of nitric oxide (NO) in biological samples. It is a cell-permeable compound that is hydrolyzed by intracellular esterases to produce the fluorescent dye DAF-FM, which reacts with NO to form a fluorescent benzotriazole derivative. The intensity of the fluorescence signal is proportional to the concentration of NO present in the sample.
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Lab products found in correlation
Te2000 u fluorescence microscope, by Nikon (1 mentions)
2 7 dichlorofluorescein diacetate h2dcf da, by Merck Group (1 mentions)
Amplex red hydrogen peroxide peroxidase assay kit, by Thermo Fisher Scientific (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Lps from e coli serotype 055 b5, by Merck Group (1 mentions)
Pherastar fs, by BMG Labtech (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Antibodies against β actin, by Santa Cruz Biotechnology (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
4 protocols using diaminofluorescein fm diacetate daf fm da
1
Fluorescence Microscopy for NO and H2O2 Visualization
2
Macrophage Nitric Oxide Production Assay
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The suspension of macrophages on 96-well plates (2 × 104 cells/well) were washed withthe PBS and treated with 180 µL/well of the tested compounds (10 μM) for 1 h and 20 µL/well LPS from E. coli serotype 055:B5 (Sigma, 1.0 μg/mL), which were both dissolved in PBS and cultured at 37 °C in a CO2-incubator for one hour. For the RNS levels measurement, 200 μL Diaminofluorescein-FM diacetate (DAF FM-DA, Sigma, final concentration 10 μM) fresh solution was added to each well, and the plates were incubated for 40 min at 37 °C, then replaced with fresh PBS, and then incubated for an additional 30 min to allow complete de-esterification of the intracellular diacetates. The intensity of DAF FM-DA fluorescence was measured at λex 485 n/λem 520 nm using the plate reader PHERAstar FS (BMG Labtech, Offenburg, Germany).
3
Quantifying Nitric Oxide in Zebrafish Larvae
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NO production in zebrafish larvae was measured by modifying the method described by Ko et al. [52 (link)]. After 7–9 hpf (hours post fertilization), the zebrafish embryos were pre-treated with 50 μM HHMP and then treated with 10 μg/mL LPS. Zebrafish larvae at 72 hpf were transferred to a 24-well plate and treated with the specific fluorescence dye diaminofluorescein-FM diacetate (DAF-FM DA; 10 μM; Sigma-Aldrich, St. Louis, MO, USA) for 3 h at 37 °C to measure NO production. The zebrafish larvae were washed three times and photographed using an LSM 700 Zeiss confocal laser scanning microscope after anesthetization with 0.03% ethyl 3-aminobenzoate methanesulfonate (MS-222; Sigma-Aldrich, St. Louis, MO, USA). The fluorescence intensity was quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).
4
Anti-inflammatory Activity Screening
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LPS, acridine orange, 2′,7′-dichlorofluorescin diacetate (DCFH 2 -DA), dimethyl sulfoxide (DMSO), and diaminofluorescein-FM diacetate (DAF-FM DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), penicillin-streptomycin (P/S), and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco/BRL (Grand Island, NY, USA). The ELISA kits for measuring prostaglandin E 2 (PGE 2 ), Fig. 1 Sample preparation and anti-inflammatory activity screening. NO generation (b) and cytotoxicity (c) of SPE and its fractions on LPSstimulated RAW 264.7 cells. NO production was measured by Griess assay and cell viability was measured by MTT assay. The data were expressed as the mean ± SE (n = 3). * p < 0.05, ** p < 0.01 as compared to LPS only-treated group and ## p < 0.01 as compared to control group interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β levels were purchased from R&D Systems Inc. (Minneapolis, MN, USA). Antibodies against β-actin, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antirabbit and anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Other chemicals and regents used in the present study were analytical grade.
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