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.
Tnf α kits
Manufactured by R&D Systems
Sourced in United States
The TNF-α kits provided by R&D Systems are designed for the quantitative determination of human Tumor Necrosis Factor alpha (TNF-α) levels in cell culture supernatants, serum, and plasma. The kits utilize a sandwich enzyme-linked immunosorbent assay (ELISA) technique to measure the concentration of TNF-α in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Griess reagent, by Beyotime (1 mentions)
Human tnf flex set, by BD (1 mentions)
Human il 1β flex set, by BD (1 mentions)
Mip 1β, by R&D Systems (1 mentions)
Human soluble protein master buffer kit, by BD (1 mentions)
Human il 8 flex set, by BD (1 mentions)
Human il 6 flex set, by BD (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
5 protocols using tnf α kits
1
Cytokine Profiling in Co-Culture Model
2
Measurement of Inflammatory Biomarkers
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Circulating levels of CRP, IL-6, and TNF-α were determined in duplicate by high sensitivity human ELISA kits. The CRP kits were obtained from Life Technologies (Fisher Scientific; Catalog #: LSKHA0031) and IL-6 (Catalog #: HS600C) and TNF-α kits (Catalog #: SSTA00E) were obtained from R&D systems (Bio-Techne Canada). The assays were performed according to the manufacturer’s instructions. The inter- and intra-assay variability was less than 15%.
3
Cell Proliferation and Cytokine Profiling
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The cells were cultured in 96-well dishes for 0, 24, 48 and 72 hours. At the desired time point, 10 ul of CCK-8 solution from the kit was added into each well. After 4 hours, the OD at 450 nM was assessed. Cytokines in the supernatant were assessed by ELISA. IL-1β, IL-6, IL-8 and TNF-α kits (R&D Systems, USA) were used to determine the IL-1β, IL-6, IL-8 and TNF-α concentrations, respectively. For the cell cycle analysis, the cells were stained with propidium iodide (PI) for half an hour. The cell cycle was assessed by flow cytometry.
4
Cytokine Profile Analysis in Serum and Lung
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For human samples, venous blood was collected and the serum was separated by centrifugation at 700 × g and stored at −80°C until analyzed. The cytokines in human serum were analyzed by CBA. Briefly, IL-1β, IL-6, IL-8, MIP-1β, TNF-α, and MCP-1 concentrations of CB workers and control workers were measured. Human Soluble Protein Master Buffer Kit, Human IL-1β Flex Set (Bead B4), Human IL-6 Flex Set (Bead A7), Human IL-8 Flex Set (Bead A9), Human MCP-1 Flex Set (Bead D8), Human MIP-1β Flex Set (Bead E4), and Human TNF Flex Set (Bead C4) were used according to the manufacturer’s protocol (BD Biosciences). Data were acquired on BD Canto and BD LSRFortessa flow cytometers and analyzed by FCAP Array Software (Soft Flow Inc., Pecs, Hungary).
For animal samples, the blood was collected from angular vein after deep anesthetization with chloral hydrate. The angular vein is a small vein near the eye. The angular vein was pierced by a needle and the blood was collected. Blood and lung homogenate were centrifuged at 700 × g and then the supernatants were collected and stored at −80°C until analyzed. IL-6, IL-8, IL-1β, MIP-1β, and TNF-α were evaluated with commercially available mouse ELISA kit according to manufacturers’ recommendations (IL-6, IL-1β, MIP-1β, and TNF-α kits were from R&D Systems, Minneapolis MN and IL-8 kit was from Cusabio Biotech Company, Wuhan, China).
For animal samples, the blood was collected from angular vein after deep anesthetization with chloral hydrate. The angular vein is a small vein near the eye. The angular vein was pierced by a needle and the blood was collected. Blood and lung homogenate were centrifuged at 700 × g and then the supernatants were collected and stored at −80°C until analyzed. IL-6, IL-8, IL-1β, MIP-1β, and TNF-α were evaluated with commercially available mouse ELISA kit according to manufacturers’ recommendations (IL-6, IL-1β, MIP-1β, and TNF-α kits were from R&D Systems, Minneapolis MN and IL-8 kit was from Cusabio Biotech Company, Wuhan, China).
5
Biochemical Assays for Diabetic Complications
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Tet (purity ≥98%) was bought from Yaji Biotechnology Co., Ltd. (Shanghai, China). The kits for hemoglobinuria, malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), creatinine (CRE) and blood urea nitrogen (BUN) were purchased from Shanghai Qiaoyu Biotechnology Co., Ltd. (Shanghai, China). The IL-10, IL-6, inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α) kits were bought from R&D Systems Co., Ltd. (Minneapolis, MN, USA). STZ was bought from Sigma (St. Louis, MO, USA).
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