In vitro anti-inflammatory activities of compounds 1-6 were evaluated by monitoring the production of NO and TNF-α in RAW264.7 cells. 16 (link) Briefly, RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 1% antibiotics and L -glutamine, and seeded in a 96well chamber (3 × 10 4 cells/well). Next, cells were treated with different concentrations of compounds 1-6 after 24 hours incubation. Then RAW264.7 cells were exposed to LPS (1 µg/mL) for another 24 hours. The concentration of NO was measured using a Griess kit (Beyotime Biotechnology, Shanghai, China), with dexamethasone as the positive control. The concentrations of TNF-α were determined by an ELISA kit (R&D company, Minnesota, USA) according to the manufacturer's instructions. Finally, the level of TNF-α was determined from a standard curve with silybin used as a positive control.
Griess kit
Manufactured by Beyotime
Sourced in China
The Griess kit is a laboratory equipment used for the colorimetric detection and quantification of nitrite ions (NO2-) in various samples. It functions by converting nitrite into a colored azo dye that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Daf fm da, by Beyotime (2 mentions)
Anti phospho p38 mapk antibody, by Cell Signaling Technology (1 mentions)
Anti cox 2 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho iκbα antibody, by Cell Signaling Technology (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Anti phospho jnk antibody, by Cell Signaling Technology (1 mentions)
Anti iκbα antibody, by Cell Signaling Technology (1 mentions)
Anti jnk antibody, by Cell Signaling Technology (1 mentions)
Anti erk antibody, by Cell Signaling Technology (1 mentions)
Anti p38 mapk antibody, by Cell Signaling Technology (1 mentions)
Anti phospho erk antibody, by Cell Signaling Technology (1 mentions)
Anti inos antibody, by Cell Signaling Technology (1 mentions)
Raw264, by National Collection of Authenticated Cell Cultures (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Solarbio (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Annexin 5 fitc pi kit, by Beyotime (1 mentions)
Donkey anti rabbit igg alexaflour 488, by Absin (1 mentions)
Atp microplate assay kit, by Absin (1 mentions)
Reactive oxygen species assay kit, by Keygen Biotech (1 mentions)
8 protocols using griess kit
1
Anti-inflammatory Evaluation of Compounds
2
NO Production in PL720-HUVEC Interaction
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To quantify the production of NO when PL720 interaction with HUVECs, a NO fluorescent probe DAF‐FM DA (Beyotime, China) was added to HUVECs solution at a final concentration of 5 µm . After incubation in the dark for 20 min in a cell culture incubator, the unloaded probes were eliminated by washing the cells three times with PBS. Next, PL720 (20 µL) was added to each well of a six‐well plate. Real‐time NO generation was monitored using CLSM (Nikon C2, Japan) to capture fluorescence images at predetermined time intervals (every 30 min for 5 h). Fluorescence was quantified using Image J software (NIH, USA). Because of the potential influence of fluorescence on cell viability and the susceptibility of fluorescence to quenching, the nitrite level in the culture supernatant was measured using a Griess kit (Beyotime, China) to indirectly assess NO content. Absorbance was measured at 540 nm using a Multiskan SkyHigh microplate reader (Thermo Scientific, USA) following the manufacturer's instructions.
3
Determination of Nitric Oxide Levels
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First, a standard curve was drawn using NO standards according to the instructions of the Griess kit (Beyotime, China). After the cells were treated with 100mg/L AGEs for 24 h, the cell supernatant of each group was extracted and added to a 96-well plate at 50 μl/well. According to the instructions, 50 μl of Griess Reagent I and 50 μl of Griess Reagent II were added to each well, respectively. Then, the absorbance was measured at 540 nm wavelength with a microplate reader and the NO concentration in the supernatant was calculated using the standard curve.
4
Anti-inflammatory Effect of Compound
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The following reagents were used in this research: Mouse macrophage cell line RAW264.7 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China); Dulbecco's Modified Eagle's Medium (DMEM) and neutral red staining solution were bought from Solarbio (Beijing, China); Lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Griess kit and ROS kit were purchased from Beyotime Biotechnology (Shanghai, China); Mouse TNF-α and IL-6 ELISA kit were purchased from Beijing 4A Biotech Co, Ltd (Beijing, China); Primer iNOS, TNF-α, IL-6 and Cox-2 were designed and synthesized by Thermo Fisher Scientific (Shanghai, China Thermo Fisher scientific (Shanghai, China); Evo M-MLV RT Kit with gDNA Clean and TB Green TM Ex TaqTM II (Tli RNadeH Plus), Bulk kit were obtained from TaKaRa; anti-NF-κB p65 antibody, anti-phospho-NF-κB p65 antibody, anti-iNOS antibody, anti-COX-2 antibody, anti-IκBα antibody, anti-phospho-IκBα antibody, anti-P38 MAPK antibody, anti-phospho-P38 MAPK antibody, anti-Erk antibody, anti-phospho-Erk antibody, anti-JNK antibody and anti-phospho-JNK antibody were bought from Cell Signaling Technology (Danvers, MA, USA).
5
Cytotoxicity Evaluation of Doxorubicin
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The polyvinylpyrrolidone (PVP, M. W. 18,000), potassium ferricyanide (K3Fe(CN)6), sodium nitroprusside (SNP), concentrated hydrochloric acid (HCl), doxorubicin (DOX), sodium dodecyl sulfate (SDS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Aladdin Company (Shanghai, China). The fluorescence probe DAF-FM DA, GreenNuc, the Annexin V-FITC/PI kit, the Calcein/PI kit, the Griess kit and Hoechst 33,342 were purchased from Beyotime Corporation (Shanghai, China). The rabbit anti-Ki67 and anti-cleaved caspase 3 primary antibodies and the anti-rabbit secondary antibody were bought from Servicebio Company (Wuhan, China).
6
Griess Assay for Nitric Oxide
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NO production was detected by a Griess kit (Beyotime Institute of Biotechnology). In short, the cell supernatants were collected and centrifuged at 1000 r/min after 24 h treatment with LPS in the presence or absence of 6-OAP. Fifty microliters of each cell supernatant were collected, equal volumes of Griess reagent I and II working solutions were added, and the absorbance at a wavelength of 540 nm was measured within 10 min using a microplate reader. Sodium nitrite was used as a standard in the assay.
7
HA-Based Hydrogel for Wound Healing
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Hyaluronic acid (HA, 90 ~ 100 kDa) was purchased from Shyuanye (Shanghai, China). EDC·HCl and CuCl2 were supported by Aladdin (Shanghai, China). NHS was obtained from Bidepharm (Shanghai, China). Rabbit anti-α-SMA, rabbit anti-FAP-α and donkey anti-rabbit IgG-AlexaFlour 488 were purchased from Absin (Shanghai, China). Neocuproine hydrochloride monohydrate was obtained from Macklin (Shanghai, China). Griess Kit, NO fluorescent probe and BCA protein assay kit were purchased from Beyotime (Shanghai, China). Cell viability (live dead cell staining) detection kit and Reactive Oxygen Species Assay kit were obtained from Keygen (Jiangsu, China). Mouse TGF-β Elisa kit and ATP Microplate Assay Kit was supported by Absin (Shanghai, China). All the antibodies were purchased from Biolegend (San Diego, CA, USA). HMGB1 Elisa kit was supported by Tongwei (Shanghai, China). Anti PD-L1 antiboby was purchased from Bio X Cell (West Lebanon, New Hampshire, USA).
8
Evaluating Nitric Oxide Release from G@Gel
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The GSNO was synthesized according to our previous studies [43 (link)]. To evaluate the release of NO from G@Gel, GSNO (0.2 mg·mL−1, 1.95%) was encased in hydrogel and added in 96-well plate mixed with GSH (10 mM). The released NO was measured by Griess Kit (Beyotime). Furthermore, hydrogel with GSNO was irradiated for different time to investigate the effect of NIR on the release of NO. The method was the same as described above.
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