The secretion levels of IFN-β in the cell supernatants of mouse BMMs and BMDCs were measured using the IFN-β DuoSet ELISA kit (R&D Systems) according to the manufacturer’s instructions. Briefly, the captured antibodies were diluted with PBS to a working concentration, added at 100 μL/well to a 96-well plate, and incubated overnight at room temperature. The next day, 100 μL/well of the diluted cell supernatant sample or standard was added and incubated at room temperature for 2 h. Then, 100 μL/well of antibody diluent was added for 2 h, followed by 100 μL/well of streptavidin-HRP diluent for 20 min. Finally, 100 μL/well of substrate solution was added and incubated at room temperature for 20 min, followed by 50 μL/well of stop solution. The OD450 and OD570 values were determined using a microplate reader (BioTek, Winooski, VT, USA).
Ifn β duoset elisa kit
Manufactured by R&D Systems
The IFN-β DuoSet ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human interferon beta (IFN-β) levels in cell culture supernatants, serum, and plasma.
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3 protocols using ifn β duoset elisa kit
1
Quantification of IFN-β Secretion in Mouse Immune Cells
2
Verifying Flagellin-Induced IFN-β Expression
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According to the results of RNA-Seq analysis, some significantly upregulated immune-related differentially expressed genes (DEGs) were selected for verification. The RAW264.7 cells were treated with 1 μg/mL endotoxin-free FliC. After 5 h of stimulation, the cells were collected for RNA extraction. The mRNA levels of the DEGs were measured using qRT-PCR. To further verify that flagellin itself had the ability to induce IFN-β, RAW264.7 cells were stimulated by FliC (1 μg/mL), FliC + Trypsin, LPS (0.1 μg/mL) and LPS + Trypsin groups. Trypsin pre-treatment was conducted by incubation of FliC or LPS with Trypsin (1:2 v/v) at 37 °C for 10 min before stimulation. DMEM and Trypsin groups were set as negative controls. After 5 h of stimulation, cells and cultural supernatants were collected, respectively. The mRNA level of IFN-β was detected by qRT-PCR, and the secretion level of IFN-β in the supernatant was detected by IFN-β DuoSet ELISA kit (R&D Systems).
3
Comprehensive Cytokine Profiling in Mouse Tissues
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Mouse spleen, hindlimb muscle (including quadriceps, tibialis anterior, gastrocnemius, and soleus muscles), cardiac muscle, serum, and white adipose tissue were collected. Tissues were homogenized using a Dounce homogenizer (100 strokes per tissue sample) on ice in prechilled buffer containing 100 mM tris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and proteinase inhibitor. The tissue lysate was centrifuged for 10 min at 13,000 rpm at 4°C. The tissue lysate supernatants, together with the mouse serum collected using sodium citrate tubes, were analyzed using the mouse IL-15/IL-15Rα uncoated ELISA Kit (88-7215-22, Life Technologies), IL-12 p40 DuoSet ELISA Kit (DY2398-05, R&D Systems), IFN-β DuoSet ELISA Kit (DY8234-05, R&D Systems), IFN-γ DuoSet ELISA Kit (DY485-05, R&D Systems), IL-6 DuoSet ELISA Kit (DY406-05, R&D Systems), and TNFα DuoSet ELISA Kit (DY410-05, R&D Systems) following the manufacturer’s instructions. The protein levels in the tissues and serum were normalized to the tissue weight and serum volume, respectively. Where indicated, IL-12 p40 in cell culture supernatants was analyzed and normalized to the protein levels of the cell pellets.
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