BMDMs were cultured in 96-well plates with a density of 8×103 cells per well. After one day, PAM was added to the cells at the specified concentrations for 24 hrs. By measuring the concentration of nitrite from the cell culture supernatants, the generation of NO could be evaluated. The nitrite generation was assessed by the Griess reaction using a nitric oxide assay kit (Applygen, Beijing, China) following the protocol provided by the manufacturer.
Nitric oxide assay kit
Manufactured by Applygen
Sourced in China
The Nitric Oxide Assay Kit is a laboratory tool designed to measure the concentration of nitric oxide (NO) in various biological samples. The kit utilizes a colorimetric method to quantify NO levels, providing researchers with a reliable and efficient means of analyzing this important signaling molecule.
Automatically generated - may contain errors
Lab products found in correlation
Indole 3 acetic acid, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Sartorius (1 mentions)
Anti nf κb p65, by Beyotime (1 mentions)
Tin protoporphyrin 9, by Cayman Chemical (1 mentions)
Ch223191, by MedChemExpress (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Hifair 2 1st strand cdna synthesis supermix for qpcr, by Yeasen (1 mentions)
Anti ho 1, by ABclonal (1 mentions)
5 protocols using nitric oxide assay kit
1
Nitric Oxide Generation in BMDMs
2
Nitric Oxide Evaluation in Media
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Before treatment with chemicals or UA, media were replaced with Dulbecco’s modified Eagle’s medium (DMEM). The supernatant or serum was centrifuged and subjected to NO level evaluation using the Nitric Oxide Assay Kit (Applygen Technologies, China) according to the manufacturer’s instructions. The end-point measured formula was NO2−..
3
Nitric Oxide Quantification Using Griess Assay
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NO production was determined using a nitric oxide assay Kit (Applygen technologies Co., Ltd., Beijing, China) based on the Griess reaction principle [14 (link),15 (link)]. A quantity of 50 microliters of culture supernatant was transferred to 96–well plates and mixed with 50 microliters of Griess R1 reagent after incubation at room temperature for 5 min. The samples were then mixed with an equal volume Griess R2 reagent for another 5 min. The absorbance of each mixture at 540 nm was measured using a microplate reader [16 (link)].
4
Indole-3-acetic acid and LPS Inflammation
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Indole-3-acetic acid and LPS (lipopolysaccharides from Escherichia coli O55:B5) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tin protoporphyrin IX (SnPP) was obtained from Cayman Chemical (Ann Arbor, MI, USA). CH-223191 was purchased from MedChem Express (Monmouth Junction, NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM) was from Biological Industries (Kibbutz Beit Haemek, Israel). Fetal bovine serum (FBS) was purchased from Hyclone (South Logan, UT, USA). Cell counting kit (CCK-8), TRIzol RNA extraction reagent, nitric oxide assay kit, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and Hoechst 33,342 were obtained from Applygen Technologies Inc. (Beijing, China). Hieff® qPCR SYBR® Green Master Mix and Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) were procured from Yeasen biotech Co. (Shanghai, China). Antibody against β-actin was from Santa Cruz Bio. (Santa Cruz, CA, USA). Anti-HO-1 was purchased from ABclonal Inc. (College Park, MD, USA). Anti-NF-κB p65 was from Beyotime (Shanghai, China). ECL Western blotting detection kit, horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG), and anti-mouse IgG were purchased from Huaxingbio. (Beijing, China). ELISA kits for IL-1β, IL-6, TNF-α, and MCP-1 were bought from ExCell Biotech (Taicang, Jiangsu, China).
5
Nitric Oxide Level Evaluation
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Before treatment with chemicals or UA, the medium was replaced with DMEM. The culture supernatant or serum of animals was centrifuged and subjected to NO level evaluation using the Nitric Oxide Assay Kit (Applygen Technologies, China) according to the manufacturer's instructions. The endpoint compound was NO2− [7 (link)].
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