DF were quiesced for 24 h and then treated with TNF-α (0–2.5 ng/ml) in DMEM + 10% FBS for 24 h. Conditioned medium was collected and the concentration of TNF-α quantified using a Human TNF-α Quantikine ELISA (R&D Systems) according to manufacturer’s instructions.
Human tnf α quantikine elisa
Manufactured by R&D Systems
Sourced in United Kingdom
The Human TNF-α Quantikine ELISA is a quantitative sandwich enzyme immunoassay designed to measure human tumor necrosis factor alpha (TNF-α) levels in cell culture supernates, serum, and plasma. It utilizes a solid-phase antibody and an enzyme-linked detection antibody to quantify the target analyte.
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3 protocols using human tnf α quantikine elisa
1
TNF-α Quantification in Conditioned Medium
2
Therapeutic Effects of hUCB-MSCs on Atopic Dermatitis
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The mechanism underlying the therapeutic effects of mesenchymal stem cells (MSCs) in atopic dermatitis (AD) is unclear. The present study demonstrates for the first time that subcutaneous administration of human umbilical cord blood-derived MSCs (hUCB-MSCs) alleviates DF-induced AD in mice by suppressing secretion of tumor necrosis factor-alpha (TNF-α) by mast cells, and that hUCB-MSCs elicit this effect by secreting TGF-β. Additionally, it was found that TGF-β secreted from hUCB-MSCs inhibits IgE secretion by activated B cells via direct and indirect pathways. These findings suggest a scientific rationale for treating allergic diseases, including AD, with hUCB-MSCs.
were collected in phosphate-buffered saline and centrifuged at 500g for 5 minutes to eliminate residual IgE. LAD2 cells were then cultured in fresh StemPro-34 medium containing 3 μg/mL anti-IgE (Millipore), IL-33, and ionomycin for 24 hours. Thereafter, LAD2 cells were collected in phosphate-buffered saline, centrifuged at 500g for 5 minutes, and cocultured with hUCB-MSCs or HEK293FT cells in StemPro-34 medium for 16 or 24 hours. Finally, the culture medium was harvested and analyzed using a Human TNF-α Quantikine ELISA (R&D Systems).
were collected in phosphate-buffered saline and centrifuged at 500g for 5 minutes to eliminate residual IgE. LAD2 cells were then cultured in fresh StemPro-34 medium containing 3 μg/mL anti-IgE (Millipore), IL-33, and ionomycin for 24 hours. Thereafter, LAD2 cells were collected in phosphate-buffered saline, centrifuged at 500g for 5 minutes, and cocultured with hUCB-MSCs or HEK293FT cells in StemPro-34 medium for 16 or 24 hours. Finally, the culture medium was harvested and analyzed using a Human TNF-α Quantikine ELISA (R&D Systems).
3
Postprandial Metabolic and Inflammatory Markers
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At each time point, 10 mL of venous whole blood was taken and dispensed into serum separation and lithiumheparin (Vacuette; Greiner Bio-One GmbH, Kremsmünster, Austria) tubes before being centrifuged for 15 min at 2000g at 4°C and stored at -80°C for retrospective analysis of triglycerides (TGs) (Serum Triglyceride Determination Kit; Sigma-Aldrich, St. Louis, MO, USA). Apolipoprotein B48 (APO B48 ; Antibodies-online, USA), non-esterified-fatty acids (NEFA) (RANBUT; Randox Laboratories, London, UK), plasma glucagon (Glucagon EIA; Sigma-Aldrich) and tumour necrosis factor alpha (TNF-α) (Human TNF-α Quantikine ELISA; R&D Systems, Roche Diagnostics, West Sussex, UK) were measured hourly. Plasma fibrinogen (ab108842, Fibrinogen Human ELISA Kit; Abcam, Japan), HTF (Human Tissue Factor activity ab108906; Abcam, UK) and PAI-1 (Human PAI-1/serpin ELISA Kit DSE100; R&D systems, UK) were measured at rest, 3 h and 6 h postmeal. The intra-assay coefficient of variation was <10% for all assays. Due to increased assay cross-reactivity with insulin detemir, only participants treated with insulin glargine were included in serum insulin analysis (n = 8).
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