EDTA-anticoagulated whole blood was collected by intracardial puncture and the assay was performed according to manufactureŕs instructions (Mouse Lipopolysaccharides (LPS) ELISA kit, Cusabio, USA).
Lps elisa kit
Manufactured by Cusabio
Sourced in China
The LPS ELISA kit is a laboratory equipment used for the quantitative detection of lipopolysaccharides (LPS) in various sample types. It is a sandwich enzyme-linked immunosorbent assay (ELISA) designed to measure LPS concentrations.
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Vancomycin, by Maclin Biochemical Technology (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
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Cd25 pe, by BioLegend (1 mentions)
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Cd38 brilliant violet 421, by BioLegend (1 mentions)
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5 protocols using lps elisa kit
1
Mouse LPS ELISA Assay Protocol
2
Inflammatory Bowel Disease Cytokine Profile Assay
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Reference standards, including caffeic acid, quercetin, isoquercitrin, ferulic acid, vitexin, vicenin II, schaftoside, isoschaftoside, rutin, hyperoside, salicylic acid, rosmarinic acid, daidzein, oleanolic acid, stigmasterol, epinodosin, methyl rosmarinate, kamebakaurin, shikokianin, lasiokaurin, oridonin, lasiodonin, ponicidin, and nodosin were purchased from Refmedic Biotechnology Co., Ltd. (Chengdu, China). Additionally, 5-ASA was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), while DSS (molecular weight: 36–50 kDa) was obtained from International Laboratory USA (San Francisco, CA, USA). The antibiotics, namely, vancomycin, neomycin sulfate, ampicillin, and metronidazole, were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Mouse serum interleukin (IL)-17, IL-4, transforming growth factor (TGF)-β1, and interferon (IFN)-γ enzyme-linked immunosorbent assay (ELISA) kits were obtained from Novus Biologicals (Littleton, CO, USA). A lipopolysaccharide (LPS) ELISA kit was provided by CUSABIO (Wuhan, China). A fixation/permeabilization solution kit (BD GolgiPlug) and CD25, Foxp3, CD4, and IL-17A antibodies used for Th17 and Treg cell phenotyping were purchased from BD Biosciences (Franklin Lakes, NJ, USA).
3
Quantifying Immune Activation Markers in HIV
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Sera samples of immunological responders and non-responders were collected to measure the bacterial translocation markers. Following the standard protocols, human Lipopolysaccharides (LPS) ELISA Kit (CUSABIO; Wuhan, China) and Human soluble CD14 (sCD14) ELISA Kit (MultiSciences, Hangzhou, China) were used to test plasma LPS and sCD14. Flow cytometry and Cobas Amplicor (Roche Molecular Systems Inc., Branchburg, New Jersey, United States) were used to quantify CD4 + /CD8 + T-cells and HIV-1 RNA, respectively. Fresh anticoagulated whole blood was used to quantify the expressing markers of immune activation (CD25 +, CD38 +, HLADR +, or CD38 + /HLA-DR +) of CD4 + and CD8 + T-cells and immune senescence (CD57 +) by BD FACS Canto II flow cytometer (BD Biosciences, California, United States). The antibodies needed during the experiment were purchased from Biolegend (San Diego, CA), including CD3-FITC, CD4- PerCP/Cy5.5, CD8-Brilliant Violet 510™, CD38-Brilliant Violet 421, CD25-PE, HLA-DR-APC/Fire™ 750, and CD57-allophycocyanin (APC).
4
Serum LPS Quantification in NSCLC
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Peripheral blood samples were collected from the NSCLC patients in BD Vacutainer® (BD Bioscience, San Jose, CA, USA) serum tubes. After the collection of the blood, the samples were allowed to clot for one hour at room temperature (23–24 °C) and, after that time, were centrifuged at 4000 rpm for 10 min. Following centrifugation, the serum was separated from the blood and transferred into sterile 1.0 mL cryotubes using a Pasteur pipette. The lipopolysaccharide (LPS) concentrations in the serum were analyzed using commercially available enzyme-linked immunosorbent assay kits (LPS ELISA kit, USACSB-E09945h, CUSABIO, Wuhan, China) according to the manufacturer’s instructions. All samples were analyzed in duplicate.
5
Isolation and Characterization of M1 Macrophage Membranes
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First, RAW264.7 cells were stimulated for 24 h with LPS (100 ng mL‒1) and IFN-γ (50 ng mL‒1) to generate M1-polarized macrophages. After washing with PBS three times, the cells were then resuspended in PBS and incubated overnight in a hypotonic lysing buffer (4 °C). Then, a tight-fitting pestle was used to grind the cells 20 times before centrifugation (20,000 g, 25 min). The supernatant was collected and centrifuged at 100,000 g for 40 min to collect the macrophage membrane. A BCA protein assay was used to determine the protein concentration of purified macrophage membranes. To further investigate whether residual LPS and IFN-γ existed on M1 macrophage membrane, the cell membrane lysates of 1 mg M1 (or M0) macrophage membranes were analyzed using LPS ELISA kit (#CSB-E13066m, Cusabio, China) according to the manufacturer’s instructions. For IFN-γ measurement, the M1 (or M0) macrophages were stained with PE anti-mouse IFN-γ antibody (1:200, Biolegend, #505807) and then analyzed by flow cytometry.
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