We synthesized 3′,4′-dihydroxyflavone and flavone (Fig. 1A ) using our previously reported procedure (An et al., 2017 (link)). LPS from Escherichia coli serotype O55:B5 (L6529) and luteolin (L9283) were purchased from Sigma-Aldrich (MO, USA). HyClone (GE Healthcare) culture media and other materials were used for cell culture. Rabbit antibodies against p38 (#9212S), phosphor-p38 (#9215S), JNK (#9252S), phosphor-JNK (#9251S), ERK (#9101L), phosphor-ERK (#9102), iNOS (#2982S) and COX-2 (#4842S) were purchased from Cell Signaling Technology (MA, USA). Antibodies against β-actin (SC-47778 HRP) and NF-κB (rabbit, SC-372) were purchased from SantaCruz Biotechnology (CA, USA). A rabbit antibody against anti-ionized calcium-binding adapter molecule 1 (Iba-1, #019-19741) was purchased from Wako (Tokyo, Japan). Secondary antibodies were purchased from Bio-Rad Laboratories (CA, USA), SantaCruz Biotechnology and Invitrogen (CA, USA). Mouse TNF-α and IL-6 ELISA kits were purchased from BD. A mouse IL-1β kit was purchased from Invitrogen.
Mouse il 1β kit
Manufactured by Thermo Fisher Scientific
The Mouse IL-1β kit is a laboratory assay used to measure the concentration of the cytokine interleukin-1 beta (IL-1β) in mouse biological samples. It provides a quantitative method for detecting and analyzing IL-1β levels.
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2 protocols using mouse il 1β kit
1
Synthesis and Anti-inflammatory Evaluation of Flavonoids
2
Quantification of IL-1β and Dual-Luciferase Assay
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The concentration of IL-1β released by A1s was detected by Enzyme-linked immunosorbent assay (ELISA) for quantitative detection of mouse IL-1β Kit (Invitrogen, ThermoFisher Scienti c). The experimental operation was performed according to the protocol of the kit. The colorimetric optical density (OD) was measured by SpectraMax®i3x enzyme labeling instrument (Molecular Devices, USA).
Dual-luciferase reporter assay HEK 293t cells were planted in a 24-well plate. For the construction of plasmids, WT or MUT Cntfr α 3'UTR fragments were inserted into PmirGLO Dual-Luciferase miRNA Target Expression Vector (BioSune, Jinan, China). Then, plasmids, miR-21a-5p mimic, inhibitor, and negative control were transfected into HEK 293t cells. After 48 hours, added 1 × PLA cell Lysis Buffer and shaken to lyse the cells at room temperature for 15min, then collected the cell lysate. 20μl cell lysate was added to a dedicated 96-well plate, then 100μl LARII and 100μl Stop & Glo®Reagent were added in turns by the Centro XS 3 LB 960 (Berthold, Germany) and MikroWin software to detect the re y luciferase activities and Renilla luciferase activities respectively. We completed this experiment using the Promega Dual-Luciferase system (Promega, Madison, USA). The difference of luciferase activity between re y and Renilla was calculated and analyzed.
Dual-luciferase reporter assay HEK 293t cells were planted in a 24-well plate. For the construction of plasmids, WT or MUT Cntfr α 3'UTR fragments were inserted into PmirGLO Dual-Luciferase miRNA Target Expression Vector (BioSune, Jinan, China). Then, plasmids, miR-21a-5p mimic, inhibitor, and negative control were transfected into HEK 293t cells. After 48 hours, added 1 × PLA cell Lysis Buffer and shaken to lyse the cells at room temperature for 15min, then collected the cell lysate. 20μl cell lysate was added to a dedicated 96-well plate, then 100μl LARII and 100μl Stop & Glo®Reagent were added in turns by the Centro XS 3 LB 960 (Berthold, Germany) and MikroWin software to detect the re y luciferase activities and Renilla luciferase activities respectively. We completed this experiment using the Promega Dual-Luciferase system (Promega, Madison, USA). The difference of luciferase activity between re y and Renilla was calculated and analyzed.
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