The joint fluid samples of rats were collected to detect the content of IL‐6, tumor necrosis factor (TNF)‐α and IL‐1β using IL‐6, TNF‐α and IL‐1β ELISA detection kits (PI328, PT516, PI303, Beyotime Biotechnology, China), respectively. The joint fluid samples were added into ELISA plates and incubated at room temperature for 2 h. After being washed 5 times with washing solution, each well of the plate was supplemented with 100 μl of diluted detection antibody, followed by the incubation at room temperature for 1 h. Thereafter, the plate was added with washing solution and rinsed 3 times, and 100 μl of diluted horseradish peroxidase‐labeled streptavidin was put into each well to incubate the plate at room temperature for 30 min. Afterward, 100 μl coloring substrate 3,3′,5,5′‐Tetramethylbenzidine (TMB) solution (P0209, Beyotime Biotechnology, China) was added to each well and incubated the plate at room temperature for 20 min in the dark, and then 50 μl stop solution was applied to stop the color reaction. Finally, the microplate reader (SPECTROstar® Nano, BMG LABTECH, USA) was used to detect the optical density (OD) value of each well at the wavelength of 450 nm and 570 nm.
Pt516
Manufactured by Beyotime
Sourced in China
The PT516 is a laboratory pH meter designed to measure the pH value of various aqueous solutions. It features a digital display for easy reading of the measured pH value.
Automatically generated - may contain errors
Lab products found in correlation
Pi303, by Beyotime (8 mentions)
Pi328, by Beyotime (6 mentions)
Spectrostar nano, by BMG Labtech (1 mentions)
Ab186031, by Abcam (1 mentions)
S0101, by Beyotime (1 mentions)
S0131, by Beyotime (1 mentions)
S0082, by Beyotime (1 mentions)
Pi525, by Beyotime (1 mentions)
Mbs9141543, by MyBioSource (1 mentions)
S0053, by Beyotime (1 mentions)
Glutathione (gsh), by Beyotime (1 mentions)
Filtermax f5, by Molecular Devices (1 mentions)
Csb e15037r, by Cusabio (1 mentions)
Elisa kit, by USCN (1 mentions)
9 protocols using pt516
1
Cytokine Quantification in Rat Joints
2
Cytokine Levels Quantification via ELISA
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3
Comprehensive Biochemical Analyses in Neuroendocrine Stress Response
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We used ELISA kits to measure NKA activity (MBS7245054, MyBioSource, San Diego, CA, USA), ADP/ATP ratio (MAK135-1KT, Sigma-Aldrich, Burlington, VT, USA), NAD(P)H activity (ab186031, Abcam, Cambridge, UK), NF-κB activity (ABIN6958236, Antibodyies, Limerick, ME, USA), and SOD activity (S0101, Beyotime, Shanghai, China). For part II, we used Norepinephrine Assay Kit (H096, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) to measure the NE levels in the plasma. The levels of oxidative stress-related indicators, including malondialdehyde (MDA, S0131, Beyotime), glutathione (GSH, S0073, Beyotime), oxidized glutathione (GSSH, S0053, Beyotime), and catalase (CAT, S0082, Beyotime) in the PVN, and pro- and anti-inflammatory cytokines, including IL-1β (PI303, Beyotime), MCP-1 (PC128, Beyotime), IL-6 (PI328, Beyotime), IL-10 (PI525, Beyotime), TNF-α (PT516, Beyotime), IL-8 (MBS9141543, MyBioSource), and IL-4 (BMS628, Thermo Fisher, Waltham, US) were also measured by ELISA kits (32).
4
Inflammatory Cytokine Profiling after SAH
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Brain tissue samples were obtained at corresponding time points after SAH. Moreover, rhwnt1, siwnt1 RNA, and anti-Frizzled1 interventions were applied once SAH models were established. Levels of IL-1β, IL-6, and TNF-α in brain tissue were measured using specific enzyme linked immunosorbent assay kits for rats (PI303, PI328, PT516; Beyotime), in accordance with the manufacturer’s instructions. Briefly, equal quantities of brain tissue from different groups were cut and ground to generate protein samples. Standard samples were diluted to different concentrations and target samples added into corresponding wells of 100 μL. Reaction wells were sealed with transparent film, and incubated at room temperature for 120 minutes. Next, prepared biotinylated antibody was added into the reaction well, and incubated for 1 hour at room temperature. Horseradish peroxidase was added to detect streptavidin, and incubated for 20 minutes at room temperature. Finally, 3,3′,5,5′-tetramethylbenzidine solution was added and incubated for 20 minutes at room temperature. After stop solution was added, absorbance values were measured at 450 nm immediately using a spectrophotometer (FilterMax F5, Molecular Devices, San Francisco, CA, USA). Values were expressed as pg/mL.
5
Cytokine and ROS Quantification
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ELISA refers to a solid phase adsorption assay based on an enzyme as a label and based on an immunological binding reaction between an antigen and an antibody. We selected ELISA to analyze the levels of Tumor Necrosis Factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and reactive oxygen species (ROS). The cells to be tested were first subjected to a 24-hr digestion process. The cell supernatant was then extracted. According to the kit instructions, TNF-α (PT516, Beyotime), IL-1β (PI303, Beyotime), IL-6 (PI328, Beyotime) and ROS (CSB-E15037r, Cusabio, Wuhan, China) were detected in the cell supernatant. Finally, the absorbance was measured by a spectrophotometer, and the content of different factors was calculated.
6
Cytokine Levels in H9C2 Cells
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7
Cytokine and Antibody Detection
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8
Quantifying Inflammatory Mediators in Lung Injury
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Interleukin (IL)‐6 ELISA kit (PI328, Beyotime, Shanghai, China) was used to measure the IL‐6 level of the lung homogenates. The optical density (OD) values of the testing samples were measured, and the level of IL‐6 in the sample was calculated from the standard curve generated by the standard samples offered within the ELISA kit. Tumour necrosis factor‐α (TNF‐α) (PT516, Beyotime) and IL‐1β (PI303, Beyotime) kit testing was also conducted in this experiment. Furthermore, the levels of intestinal mucosal injury markers (i‐FABP) and diaminoxidase (DAO) in portal vein blood were determined by an ELISA kit supplied by Wuhan USCN Business, according to the manufacturer's instructions.
9
Spinal Cord Cytokine Quantification
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Fourteen days after SNI surgery, the samples were collected from the lumbar spinal cord to determine the levels of TNF‐α (Beyotime Biotechnology, Shanghai, China; PT516), IL‐1β (Beyotime Biotechnology, Shanghai, China; PI303), and IL‐6 (Beyotime Biotechnology, Shanghai, China; PI328). The specific experimental operation was carried out according to the instructions.
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