RAW 264.7 cells were seeded into 24-well plates with the density of 100,000 per well and cultured overnight. Next day, cells were incubated with LPS or DMF as indicated. Cell supernatants were collected and added to 96 well plates. 50 μL samples were co-incubated with 50 μL Griess Reagent Ⅰ and 50 μL Griess Reagent Ⅱ (Beyotime, Shanghai, China) for 15 min at room temperature (20 °C ± 1 °C). The amount of NO production was monitored at 540 nm by an Infinite 200 PRO plate reader.
Griess reagent
Manufactured by Beyotime
Sourced in China
Griess reagent is a laboratory chemical used for the detection and quantification of nitrite ions (NO2-) in aqueous solutions. It is a colorimetric assay that produces a pink or purple-colored azo dye in the presence of nitrite ions. The Griess reagent is a commonly used analytical tool in various fields, including biochemistry, environmental analysis, and clinical diagnostics.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Collagenase type 2, by Merck Group (4 mentions)
Absolute ethyl alcohol, by Tianjin Damao (3 mentions)
Sodium hydroxide (naoh), by Tianjin Damao (3 mentions)
N hexane, by Tianjin Damao (3 mentions)
Elisa kits of pge2, by R&D Systems (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
Sybr green master mix, by Bio-Rad (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Nano2, by Beyotime (2 mentions)
Biotinylated secondary antibody, by Vector Laboratories (2 mentions)
Acetonitrile, by Merck Group (2 mentions)
Hydrogen peroxide assay kit, by Beyotime (2 mentions)
E coli 0111 b4, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
110 protocols using griess reagent
1
Nitric Oxide Production in RAW 264.7 Cells
2
Synthesis of Multifunctional Nanoparticles
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FeCl3·6H2O, sodium oleate, oleic acid were purchased from Tianjin Yongda Chemical Reagent Co., Ltd.; oleylamine (80–90%) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. Cetyl trimethyl ammonium bromide (CTAB) and ammonium nitrate were purchased from Beijing Chemical Reagent Co., Ltd.; n-hexane, absolute ethyl alcohol and sodium hydroxide (95%) were purchased from Tianjin Damao Chemical Reagent factory; octadecene, N-(2-aminoethyl)-3-amopropyl trimethoxysilane (EDS), tetraethoxysilane (TEOS) were purchased from Beijing Balinwei Reagent Co., Ltd.; Polyoxyethylene-polypropylene-polyoxyethylene copolymer (F127) was purchased from Germany BASF Company; chloroauric acid (HAuCl4·3H2O, 99.99%) was purchased from Alfa Aesha (China) Chemical Co., Ltd.; folic acid (FA, ≥97%), doxorubicin hydrochloride (DOX, ≥98%) and S-Nitrosylglutathione (GSNO) was obtained from Aladdin Bio-chem Technology Co., Ltd.; Griess reagent Shanghai Biyuntian Biotechnology Co., Ltd. 1-hydroxybenzotriazole (HOBT) and O-benzotriazole - tetramethyl urea hexafluorophosphate (HBTU) were purchased from Gill Biochemical (Shanghai) Co., Ltd. All chemicals were analytical reagent grade and used without further purification.
3
Nitric Oxide Quantification via Griess Assay
4
Microglia Nitric Oxide Regulation
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BV2 and primary microglia seeded in 24-well plates were pre-treated with Pon for 2 h and stimulated with LPS (0.5 µg/mL for BV2 cells and 0.1 µg/mL for primary microglia) for 24 h. The concentrations of NO in the supernatants were detected using the Griess reagent (Beyotime Biotech, Nantong, China). The OD value was measured at 540 nm with a microplate reader.
5
Nitric Oxide Quantification in LPS-Stimulated RAW 264.7 Cells
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The amount of NO in the culture medium was determined by Griess reagent (Beyotime, Shanghai, China). RAW 264.7 cells were seeded in a 12-well plate (3 × 105 cells/each well) and incubated at 37 °C with 5% CO2 for 24 h. After that, the cells were pretreated with 5-HMF at various concentrations (0, 31.5, 63.0 and 126.0 μg/mL) for 6 h and afterward stimulated with LPS (1 μg/mL) for 18 h. Then, 50 μL of a supernatant medium from each well was transferred into a 96-well plate and subsequently, Griess I reagent (50 μL) and Griess II reagent (50 μL) were added to each well. The absorbance was measured at a wavelength of 540 nm using the Modular multitechnology microplate reader. Two controls (a blank control and a normal control) were prepared as follows: The blank control was a sample group without 5-HMF pretreatment and LPS stimulation; the normal control was a sample group without 5-HMF pretreatment but with LPS stimulation.
6
Measuring Nitric Oxide in Alveolar Macrophages
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Primary mouse alveolar macrophages (5 × 10 5 cells) were pretreated with rmPK2 for 16 hours and then challenged with P. aeruginosa at a MOI ratio of 1:100 at 37 °C for indicated times. Nitric oxide (NO) is inherently unstable and quickly metabolizes into nitrate/nitrite in cells. NO levels were determined by assaying the levels of nitrate/nitrite using the Griess reagent (Sigma-Aldrich, MO, USA). The cell supernatants and standards (NaNO 2 , Beyotime, Shanghai, China) were mixed with Griess reagent (1:1) for 15 minutes and then measured at 540 nm.
7
Nitric Oxide Quantification in BV-2 Cells
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BV-2 microglial cells were purchased from Peking Union Medical College Cell Bank (Beijing, China) and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, penicillin G (100 U mL−1), and streptomycin (100 μg mL−1) at 37 °C in a humidified incubator with 5% CO2. The NO concentration was detected by the Griess reagent.33 (link) Quercetin was used as a positive control. BV-2 cells were seeded at the density of 1.5 × 105 cells per mL in 96-well culture plate and treated with each compound and LPS (1.0 μg mL−1) for 24 h. After that, 50 μL of cell-free supernatant was allowed to react with an equal volume of Griess reagent (Beyotime Institute of Biotechnology, Shanghai, China) for 15 min at room temperature in the dark. Then, the absorbance was measured at 540 nm using a microplate reader. The cell viability of the cultured cells was detected by MTT-based colorimetric method.
8
Neuroprotective Effects of Icariin in 6-OHDA Model
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Icariin (ICA, purity 98%) was bought from Nanjing Zelang Medical Technology, Co., Ltd. (Nanjing, China). 6-OHDA (CAS 28094157) was from Sigma Chemical, Co. (St. Louis, MO, United States). LPS (Escherichia coli strain O111:B4) was purchased from Calbiochem (San Diego, CA, United States). Anti-Tyrosine Hydroxylase (TH) antibody, Anti-CR3 complement receptor (OX-42), Anti-Iba1 antibody, Goat Anti-Mouse IgG H&L (TRITC), and Goat Anti-Rabbit IgG H&L (TRITC) were bought from Abcam (Cambridge, MA, United States). NF-κB signaling pathway antibodies were the products of Cell Signaling Technology (Beverly, MA, United States). Biotinylated secondary antibodies and vectastain avidin–biotin complex (ABC) kits were purchased from Vector Laboratories (Burlingame, CA, United States). ELISA kits were purchased from R&D Systems (Minneapolis, MN, United States). Griess reagent was obtained from Beyotime Biotechnology (Shanghai, China).
9
Nitric Oxide Production in Macrophages
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RAW264.7 macrophages were maintained in the RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin/streptomycin) and grown in the presence of 5% CO2 and 95% O2 at 37 °C. The macrophages (5 × 104/well) were seeded in a 96-well plate for 24 h and pre-treated with 2c or HSS (5, 10, and 20 μM) for 2 h before 24 h of stimulation with LPS (1 μg/mL). The Griess reagent (Beyotime, Shanghai, China) was then added to the culture supernatants, and its absorbance was measured using the SpectraMax-M3 microplate reader (540 nm, Molecular Devices, San Jose, CA, USA) to determine the NO levels.
10
Cytokine Profiling in Co-Culture Model
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The levels of TNF-α, IL-1β and IL-6 in the cell culture medium supernatants of co-culture system were detected with appropriate enzyme linked immunosorbent assay (ELISA) kits per manufacturer’s instructions. TNF-α kits was purchased from R&D System (Minneapolis, MN, USA), IL-1β and IL-6 kits were purchased from Genetimes Technology (Shanghai, China). The level of NO in supernatants was measured by Griess reagent. Griess reagent was obtained from Beyotime Biotechnology (Shanghai, China). The concentration of soluble cytokines in each sample was determined from a standard curve generated by using positive controls provided in the kits.
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