Lps eb

Female Hooded Lister rats (3–4 months old, 220g–320g) kept at a 12-h light/dark cycle environment at a temperature of 22 ​°C with access to food and water ad libitum were housed in polycarbonate cages (n ​= ​3 per cage) in the Biological Services Unit at the University of Sheffield. Animals were fed conventional laboratory rat food. Sixteen animals were randomly assigned to one of two groups (control n ​= ​8 or LPS, n ​= ​8). Haemodynamic data were acquired in all treatment groups at both 4 and 6 ​h after LPS/vehicle administration, to characterise any effects of the acutely induced LPS inflammatory response (Fig. 1).Fig. 1

Experimental timeline. Graphical representation of study design and timeline from non-recovery general anaesthesia to tissue collection. Time (minutes or hours) is given from time of injection (0 ​min).

Fig. 1Each animal received an intraperitoneal injection based on condition. Control animals were administered a saline vehicle (1 ​ml/kg), LPS animals received a dose of 2 ​mg/kg LPS-EB (lipopolysaccharide from E.coli, 0111:B4 stain-TLR4 ligand, InvivoGen, Europe) dissolved in endotoxin-free water (InvivoGen, Europe), following loss of consciousness from anaesthesia.

For whole blood stimulation, TruCulture tubes (RBM) containing Poly:IC (20 μg/mL), R848 (1 μM), LPS-EB (ultrapure) (10 ng/mL) (all Invivogen), and IFN-α2 (Intron A, Merck) dissolved in 2 mL of buffered media were batch produced as previously described^{38 (link)}. Tubes were thawed at room temperature and 1 mL of fresh blood was distributed into each tube within 15 min of collection. Tubes were mixed by inverting them and incubated at 37 °C for 22 h in a dry block incubator. After the incubation time, a valve was manually inserted into the tube to separate the supernatant from the cells. The supernatant was collected, aliquoted, and immediately stored at −80 °C for protein secretion analysis. Cell pellets of the TruCulture tubes were resuspended in 2 mL of Trizol LS (Sigma) and tubes were vortexed for 2 min at 2000 rpm and stored at −80 °C for gene expression analysis.

HA-tagged human TLR4 gene transfected stable HEK-293 cell line was purchased from Invivogen (Catalog # 293-htlr4ha). We maintained the hemagglutinin (HA)-tagged TLR4 human embryonic kidney (HEK) 293 cells in Dulbecco’s modified Eagle medium (DMEM with) added 10% fetal bovine serum, 1% penicillin/streptomycin, and antibiotics (50 µg/mL hygromycin and 10 µg/mL blasticidin) in a humidified condition of 5% CO₂ at 37 °C. Eight experiment sets were prepared with three replicates in each of them.
We treated the cells with 10 µM simvastatin (Sigma-Aldrich now Merck & Co., Inc., Kenilworth, NJ, USA) for 24 h, then stimulated them with 1 µg/mL lipopolysaccharides (LPS-EB, InvivoGen, San Diego, CA, USA) for 1 h in the freshly supplied medium. After collecting the cells, we treated the cells with our in-house ETD cross-linker (XL, 1 µmol/mL) for 30 min, followed by stopping the XL reaction with 50 mM Tris-HCl, pH 8.0. Similarly, we treated cells with simvastatin for 24 h or lipopolysaccharides for 1 h, followed by treatment with an ETD cross-linker. Moreover, we prepared control cell lines with or without the treatment of ETD cross-linker. Then, we performed IP pull-down for proteomics studies, as before [38 (link)]. Briefly, cells were lysed with IP lysis buffer containing a protease inhibitor, followed by sonication, incubation, and centrifugation at 20,000× g.

The bacterial cell wall component LPS (InvivoGen, LPS-EB) was administered to the culture medium of MEF, HUVEC, and SH-SY5Y cells (n = 3–6,) at 0.1 μg/ml for 24 h. Cells were collected for RNA extraction.

Mouse bone marrow-derived DC (BM-DC) from wild-type BALB/c and TLR4-/- mice were prepared as previously described [34 (link)]. Briefly, the bone marrow precursors were isolated from femur and tibia of respective mice. Cells were cultured at 4x10⁵/ml in bacteriological Petri dishes in 10 ml culture medium with GM-CSF (20 ng/ml; Sigma-Aldrich). Fresh medium was added at day 3 and 6 and BM-DC were used on day 8 of culture. BM-DC (10⁶ cells/well) were stimulated with 100 μg/ml of ST1 or R03 diet extracts for 18 h. As controls, BM-DC were incubated with ultrapure LPS (LPS-EB, 1 μg/ml, InvivoGen, USA). Levels of IL-10, IL-12p70, TNF-α and IL-6 in culture supernatants were determined by ELISA Ready-Set-Go! kits (eBioscience, USA) according to manufacturer’s instructions.