To determine whether WECC administration decreased the levels of TNF or IL-1β in the spinal cord, the levels of each cytokine were measured by enzyme linked immunosorbent assay (ELISA). Isoflurane anesthetized animal were transcardially perfused with 0.1 M PBS, the L4/L5 segments of the spinal cord were exposed from the vertebral column via laminectomy and identified by tracing the dorsal roots from their respective dorsal root ganglia (DRG). Collected tissues were stored in 1 mL RIPA buffer (Thermo Scientific) containing protease inhibitor cocktail (Roche, Basel, Switzerland). Samples were assayed using a commercial rat TNF ELISA kit (BD OptEIA Set Rat TNF, BD biosciences, San Jose, CA, USA) and mouse IL-1β ELISA kit (BD OptEIA Set mouse IL-1β, BD biosciences) following the manufacturer’s protocols. Optical density (O.D.) was measured at 450 nm with λ correction of 570 nm using a microplate reader (Tecan). Total amounts of protein in samples were measured using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Samples and standards were run in duplicate. All results were normalized to the total amount of protein in each sample.
Opteia mouse il 1β set
Manufactured by BD
Sourced in United States
The OptEIA Mouse IL-1β Set is a laboratory equipment product designed for the quantitative measurement of mouse interleukin-1 beta (IL-1β) levels in biological samples. The set includes the necessary components to perform this analysis.
Automatically generated - may contain errors
Lab products found in correlation
Duoset elisa kit, by R&D Systems (2 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Tecan (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Human il 1β duoset, by R&D Systems (1 mentions)
Tnfα duoset elisa kit, by R&D Systems (1 mentions)
6 protocols using opteia mouse il 1β set
1
Spinal Cord Cytokine Quantification
2
Mucosal Immune Response to Mycoplasma
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XS106 cells were added to a 24-well plate at 3 × 10 5 cells per well in 500 μL XS medium and incubated at 37°C for 16 h at a 5% CO 2 atmosphere. The cultures were centrifuged at 400 × g for 5 min, and the cells were washed with supplement-free RPMI 1640 basal medium. The cells were resuspended in 300 μL RPMI 1640 basal medium and incubated at 37°C for 24 h with intact or heat-killed M. salivarium and M. pneumoniae cells (0, 36.5, or 365 μg protein). The amounts of IL-1β in cell culture supernatants were measured by an ELISA kit (BD OptEIA™ SET Mouse IL-1β, BD Biosciences, San Jose, CA, USA). The cell culture supernatants were subjected to SDS-PAGE to distinguish mature IL-1β from pro-IL-1β, and proteins were transferred to a nitrocellulose transfer membrane and reacted with the appropriate antibody against IL-1β (R&D Systems, Minneapolis, MN, USA).
3
Quantification of Secreted Cytokines
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Secreted cytokines were quantified by enzyme linked immunosorbent assay (ELISA) using the experiments supernatants after appropriate dilution in growth media. All cytokines were measured according to the manufacturer’s instructions. For IL-1β the kit OptEIA Mouse IL-1β Set (BD Biosciences) and Human IL-1β DuoSet (R&D Systems) were used. For IL-18 the kit mouse IL-18 ELISA (MBL International) was used.
4
Measuring Cytokine Secretion in BMDM and Tissue
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Secreted cytokines were measured in the culture supernatants of the BMDMs treated per an MCC950 cytotoxicity assay or in the extracellular space of fresh tissue (capsule around subcutaneous discs) explanted from mice. All cytokines were measured according to the manufacturer’s instructions. For IL-1β, the OptEIA Mouse IL-1β Set (BD Biosciences) was used. For TNF-α, the DuoSet ELISA Kit (R&D Systems) was used. ELISA was not conducted on nerve tissue due to an insufficient amount of tissue harvested in this implantation model.
5
Cytokine Secretion Measurement
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Secreted cytokines were measured in the culture supernatants. All cytokines were measured according to the manufacturer's instructions. For IL-1β OptEIA Mouse IL-1β Set (BD Biosciences) was used and TNF-α using the DuoSet ELISA kit (R&D Systems).
6
Cytokine Quantification in Cell Cultures
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All cytokines were measured according to the manufacturer’s instructions. The OptEIA Mouse IL-1β set (559603, BD Biosciences, Wokingham, Berkshire, UK) and the TNFα DuoSet ELISA kit (DY410, R&D Systems, Abingdon, Oxfordshire, UK) were used to quantify cytokines secreted in cell culture supernatants. Serum and lung homogenates were screened by a custom flow cytometric multiplex bead array (Mouse Th1/Th2 10 plex kit FlowCytomix BMS820FF and Mouse Chemokine 6 plex kit FlowCytomix BMS821FF, Luminex Multiplex Assay, ProcartaPlex, ThermoFisher Scientific, Basingstoke, Hampshire, UK).
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