The content of NO was measured by a NO detection kit (Nanjing Jiancheng Bio Institute), which conducted following the manufacturer's protocol. NO reacts with oxygen and water is converted into nitrate and nitrite, which can change to a pink azo compound in a nitrate chromogenic agent. NO levels were determined by measuring the optical density of colored formation at 550 nm with a microplate reader. NO content was calculated based on cellular protein concentration, with sodium nitrite to generate the standard curve. Cellular NO content is expressed µmol/g cellular protein.
No detection kit
Manufactured by Nanjing Jiancheng
Sourced in China
The NO detection kit is a laboratory equipment designed to measure the concentration of nitric oxide (NO) in various samples. It provides a reliable and accurate method for quantifying NO levels, which is essential for various research and analytical applications.
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24 protocols using no detection kit
1
Nitric Oxide Quantification Assay
2
Measuring Inflammatory Mediators in Microglial Cells
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The level of NO was assessed indirectly by measuring its end-products, nitrate and nitrite. BV2 cells were seeded into 6-well culture dishes (4 × 105 cells per well) and cultured overnight. Then, BV2 cells were pre-treated with ISL for 2 h, then co-treated with 5 μM AβO for 24 h. After treatment, the supernatant was collected and measured according to the instructions of the NO detection kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China).
BV2 cells were plated in 6-well culture plates (4 × 105 cells per well) in the culture medium. Then the cells were pre-treated with ISL for 2 h and co-treated with 5 μM AβO for 24 h. Next, we used the commercial enzyme-linked immunosorbent assays (ELISA) kits (CUSABIO Technology, Wuhan, China) to measure the release of IL-1β, IL-6, and TNF-α in the cell supernatant according to the manufacturer’s protocol.
BV2 cells were plated in 6-well culture plates (4 × 105 cells per well) in the culture medium. Then the cells were pre-treated with ISL for 2 h and co-treated with 5 μM AβO for 24 h. Next, we used the commercial enzyme-linked immunosorbent assays (ELISA) kits (CUSABIO Technology, Wuhan, China) to measure the release of IL-1β, IL-6, and TNF-α in the cell supernatant according to the manufacturer’s protocol.
3
Cardiovascular Biomarker Measurement
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NO levels were assessed using NO detection Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. ET-1, Ang II, BH4, ETA, N-terminal probrain natriuretic peptide (NT-ProBNP), asymmetric dimethylarginine (ADMA), and eNOS content in serum or cardiac tissues were assessed by ELISA Kit (Jiangsu Zeyu Biological Technology Co., Ltd, Yancheng, China.) according to the manufacturer's instructions.
4
Nitric Oxide Measurement in HUVECs
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The NO content in HUVECs/si-NC and HUVECs/siCav-1 treated with or without LPS and/or Ach was also investigated. The culture supernatants were collected from all eight experimental groups following treatment. Next, NO concentrations were measured using a nitrate reductase method with an NO detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol.
5
Quantification of Inflammatory Markers
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All samples were frozen at −80 °C until all biological replicates have been acquired. Then, detection for Cytokines, ROS and NO were performed immediately. The concentration of TNF‐α (R&D Systems, MN), MMP9 (Boster, Wuhan, China), and IL‐1β (R&D Systems, MN) were detected using an ELISA as per manufacturer's instructions. Absorbance was read at 490 nm on a spectrophotometer, and sample concentrations were calculated using an equation generated from a standard curve. ROS was determined by FACS using the total ROS Assay Kit 520 nm (Affymetrix, 88‐5930) as per manufacturer's instruction. NO was measured by nitrate reductase method using NO detection kit (Jiancheng Bioengineering, Nanjing, China). Briefly, the absorbance was read at 550 nm on a spectrophotometer, and the content of NO2− is determined colorimetrically.[90 ]
6
Nitric Oxide Quantification Protocol
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NO levels in the culture supernatant and rat serum were determined using an NO detection kit (Nanjing Jiancheng Bioengineering Institute) according to the instructions. The samples and reagents 1 (R1) and R2 were mixed, and then incubated for 60 min at 37 °C. R3 and R4 were then added, the solution was mixed for 30 s, incubated at room temperature for 40 min, and then centrifuged for 10 min at 3,500 rpm. Next, 0.5 ml supernatant was removed, mixed with a chromogenic agent, and incubated for 10 min at room temperature. The colorimetric result was obtained at a wavelength of 550 nm and optical diameter of 0.5 cm. The optical density was measured, and the NO content was calculated.
7
Nitric Oxide Detection in HUVECs
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Nitrate reduction was used as an indirect measure of nitric oxide formation [36] (link), [37] (link). Briefly, HUVECs were treated with TSH (2 μmol/L) or vehicle for 24 h. After the incubation period, the supernatant was collected to detect NO secreted by HUVECs via nitrate reduction according to the instructions of the NO detection kit (Nanjing Jiancheng, Nanjing, China). A microplate reader (Molecular Devices, USA) was used to examine the OD values at 550 nm. The calculation of NO content was according to the manufacture’s instruction.
8
Cytokine and Mediator Quantification in RAW 264.7 Cells
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The secretion of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and PGE2 in the RAW 264.7 cells were assessed using the corresponding ELISA Kits (BD Bioscience, CA; R&D Systems, Minneapolis, MN). RAW 264.7 cells in each group (8 × 105) were centrifuged. The cell-free supernatants were harvested and concentrated by using an ultrafiltration cube (Millipore, Shanghai, China). Then, the pro-inflammatory cytokines and mediators in the supernatants were assessed by using specific ELISA Kits. Each sample was tested twice, and the A values were recorded at 450 nm and 570 nm. According to the A values (A450–570), standard curves were created by using the standards in Curve Expert 13.0. The quantities of these cytokines secreted by RAW 264.7 cells were calculated from each of the standard curves. NO concentration was measured using an NO detection kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions.
9
Vascular Biomarker Profiling in Cardiac Tissue
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Blood was obtained from the right carotid artery with a polyethylene catheter at the time of sacrifice. The level of NO in serum was measured using the NO Detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. ET-1, PGI2 and TXA2 levels were measured using commercial radioimmunoassay kits (Eastern Asia Radioimmunity Research Institute, Beijing, China) according to the manufacturer’s instructions. The hydroxyproline content in the hearts was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions.
10
Nitric Oxide Quantification Protocol
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Medium samples were collected from each of the samples and frozen at -80°C. NO content was assayed according the instruction for NO detection kit (Nanjing Jiancheng, China). NO3- was deoxidized to NO2- which then became color compound through Griess diazotization reaction. The absorbance was measured at 550 nm with a spectrophotometer.
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