Levels of Endotoxin-core antibodies IgM (Hycult Biotech, Uden, Netherlands), soluble CD14 (sCD14) (R&D Systems, Minneapolis, MN, United States), and internal fatty acid-binding protein (iFABP) (MyBioSource, San Diego, CA, United States) in plasma samples were measured using an enzyme-immunoassay technique (ELISA) following the manufacturer’s protocol. Plasma levels of free bacterial endotoxin were measured using a chromogenic Limulus amebocyte lysate assay (Hycult Biotech, Uden, Netherlands) following the manufacturer’s protocol. Plasma levels of circulating immune factors were measured using a Magnetic 33-plex assay (R&D Systems, Minneapolis, MN, United States) and a MAGPIX instrument (Luminex, Austin, TX, United States). Bubble plots were generated using ggplot2 R package with concentration (pg/ml).
Limulus amebocyte lysate assay
Manufactured by Hycult Biotech
Sourced in Netherlands
The Limulus amebocyte lysate (LAL) assay is a sensitive and specific test used to detect and quantify the presence of endotoxins, also known as lipopolysaccharides (LPS), in a variety of samples. The assay utilizes the reagent extracted from the blood cells (amebocytes) of the horseshoe crab, Limulus polyphemus. When exposed to endotoxins, the LAL reagent undergoes a series of enzymatic reactions that result in the formation of a visible gel or color change, which can be measured to determine the endotoxin concentration in the sample.
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3 protocols using limulus amebocyte lysate assay
1
Quantifying Endotoxin-Related Immune Markers
2
Quantifying Endotoxin-Related Immune Markers
3
Serum and Hepatic Lipid Profiling in Mice
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Blood samples were allowed to stand for 1 h at room temperature and centrifuged at 4000 rpm for 20 min to separate the upper serum. Serum lipopolysaccharide (LPS) was measured by the Limulus Amebocyte Lysate assay (Hycult Biotech). Serum levels of triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured by commercial assay kits (Nanjing Jiancheng). All measurements were performed in accordance with the manufacturer’s instructions.
Hepatic TGs were quantified using Folch extraction (18 (link)). Liver samples from mice were sectioned to chloroform-methanol (2:1) to extract lipids. The organic extract was then dried and reconstituted in isopropanol. The hepatic TG levels were determined using the assay used for determining the serum TG levels. The concentration of hepatic FGF21 was measured using a Mouse FGF-21 ELISA Kit (Immunodiagnostics Limited). All measurements were performed in accordance with the manufacturer’s instructions.
Hepatic TGs were quantified using Folch extraction (18 (link)). Liver samples from mice were sectioned to chloroform-methanol (2:1) to extract lipids. The organic extract was then dried and reconstituted in isopropanol. The hepatic TG levels were determined using the assay used for determining the serum TG levels. The concentration of hepatic FGF21 was measured using a Mouse FGF-21 ELISA Kit (Immunodiagnostics Limited). All measurements were performed in accordance with the manufacturer’s instructions.
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