Lipopolysaccharide lps escherichia coli serotype 055 b5

Lipopolysaccharide (LPS) (Escherichia coli serotype 055:B5), palmitic acid (PA) and oleic acid (OA) were ordered from Sigma (Deisenhofen, Germany). Recombinant tumor necrosis factor (TNF), interleukin (IL)-6 and leptin, and mouse Tgfb DuoSet ELISA were from R&D Systems (Wiesbaden-Nordenstadt, Germany). Ccn2 ELISA was from Hölzel Diagnostika Handels GmbH (Köln, Germany).

According to our previous research (Tang et al., 2022 (link)), RRTP is extracted from RRT fruit and preserved in microbiology and biochemical pharmaceutical engineering research center of Guizhou Medical University. Lipopolysaccharide (LPS, Escherichia coli serotype 055:B5) was purchased from Sigma-Aldrich. Acetonitrile and methanol (UPLC-grade) were collected from Merck.

Buffy coats from healthy donors were obtained after written informed consent (Sanquin Blood Bank, Nijmegen, the Netherlands). Human peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation over Ficoll-Paque (GE Healthcare). Percoll isolation of monocytes was performed as previously described as yielding a level of T cell contamination, measured by fluorescence-activated cell sorting of only 5% [13 (link), 17 (link)]. Purified monocytes were cultured in RPMI 1640 Dutch-modified culture medium (RPMI medium, Invitrogen) supplemented with 50 μg/mL gentamicin (Centraform), 2 mmol/L Glutamax (Invitrogen), 1 mmol/L pyruvate (Invitrogen), and 10% pooled human serum. Stimuli and inhibitors used were Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich, 10 ng/mL), Pam3Cys (EMC Microcollections, L2000, 10 μg/mL), 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO, Sigma-Aldrich), and 2-deoxy-d-glucose (2-DG, Sigma-Aldrich). Low-density lipoprotein was isolated from pooled human serum by ultracentrifugation and oxidized by incubating with 20 μmol CuSO₄/L for 16 h at 37 °C followed by dialysis, as previously described [4 (link)].