E coli lps

Bone marrow derived macrophages (BMDM) were prepared as previously described52 (link). BMDM were stimulated with 500 ng/ml E. coli LPS (Invivogen, CA USA) and 3 h later treated with either 5 mM ATP (Sigma, MO USA), 10 μM nigericin (Enzo Life Science, INC., NY USA), or 130 μg/mL alum (Sigma) stimulation for NLRP3 inflammasome, 400 ng/ml poly(dA:dT) (Invivogen) for AIM2 inflammasome, and Salmonella Typhimurium IR715 and ΔfljB/fliC (MOI 5) for NLRC4 inflammasome activation34 (link). BMDM were treated with Quercetin, naringenin, or silymarin at the doses of 20 and 100 μM, 30 min before the signal-2 (ATP, nigericin, e.t.c.) stimulation. Nlrp3^A350/A350 BMDM were primed with LPS (500 ng/mL) and treated with Quercetin 1 hour after priming. Supernatants were collected and assessed for IL-1β and TNF-α concentration by ELISA (eBiosciences).
Quercetin, naringenin, and silymarin were purchased from Sigma. For in vitro experiments, a stock solution of the flavonoids dissolved in 100% dimethyl sulphoxide (DMSO) at the concentration of 200 mg/ml was further diluted with culture medium prior to administration in cell cultures. For in vivo treatment, Quercetin (100 mg/kg) was first dissolved in 100% DMSO and then diluted with PBS to a final volume of 100 μl/10 g of body weight.

HEK 293T cells were seeded in a 96‐well plates at 2 × 10⁴ cells/well overnight, and cells were transiently transfected with FuGENE^® 6 (Promega) for 24 h for a total of 0.4 μg of DNA consisting of 50 ng TLR plasmids, 200 ng of pBIIXLuc reporter plasmid, 5 ng of control Renilla luciferase (pRL‐null, Promega) and 50 ng of myc‐PumA expression vector. The total amount of DNA was kept constant by adding empty vector. Where indicated, cells were treated with E. coli LPS (1 μg/ml) and Flagellin FLA‐ST (1 μg/ml), all obtained from InvivoGen, for 6 h. In the case of IL‐1β and TNFR, endogenous receptors were stimulated with IL‐1β (100 ng/ml) and TNFα (100 ng/ml), respectively. Cells were then lysed and luciferase activity measured using Dual‐Glo Luciferase Assay System (Promega).

Rainbow trout head kidney macrophages were transiently transfected for 12 h using Lipofectamine™ 3000 Transfection Reagent (Life, Shanghai, china), according to the manufacturer’s instructions, with 0.3 μg of DNA, including 50 ng of TLR-4 and TLR-2 plasmids (Miaoling Bioscience & Technology Co., Ltd., Wuhan, China), 200 ng of pBIIXLuc reporter plasmid, and 50 ng of the FLAG-stir-2 expression vector. The total amount of DNA was kept constant by adding empty vector. Where indicated, cells were treated with E. coli LPS (Invivogen) and Pam2CSK4 (Invivogen) for 8 h and lysed. Luciferase activity was measured using the Dual-Glo^® Luciferase Assay System (Promega, Beijing, china). The stir-2 sequences from SC09 genomic DNA were ligated into pCMV-Tag 2B using Exnase II (ClonExpress II, Vazyme, Nanjing, china).

IHMC monocytes were purified using CD14+ Microbeads (Miltenyi) and cultured for 24h in the presence of TLR ligands (TLR2: 10⁸/ml heat-killed Listeria monocytogenes, TLR3: 10 µg/ml polyI:C, TLR4: 1 µg/ml E. coli LPS, TLR5: 1 µg/ml B. subtilis flagellin, TLR8: 5 µg/ml ssRNA40; Invivogen). Supernatants were collected and measured for the accumulation of 12 different cytokines and chemokines using Cytometric Bead Arrays (BD Biosciences).

Isolated monocytes were plated at 1x10⁵ cells/well in 96-well plates in 200 μL of complete culture medium (RPMI 1640 with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.29 mg/mL L-glutamine). Monocytes were treated with E. coli LPS (Invivogen) at 100 ng/mL. After 18 hours, the plates were briefly centrifuged, and cell-free supernatant was harvested for analysis. TNFα and IL-6 were measured in culture supernatant by ELISA (R&D Systems) according to the manufacturer’s instructions.