According to LPS (CSB-E09945h, CUSABIO, China), IL-1β (CSB-E08053h, CUSABIO, China), TNF-α (CSB-E04740h, CUSABIO, China), and IL-6 (CSB-E04638h, CUSABIO, China) ELISA instructions, we detected LPS level in feces and placental tissues and IL-6, IL-1β, and TNF-α levels in cells. 20 mg of feces and placental tissue was taken, respectively, and the blood stains were washed with 1 × PBS. Feces and placental tissue were cut into small pieces and put into tissue grinder (homogenate tube), 200 μL 1 × PBS was added to make homogenate, then put in -20°C overnight. After repeated freeze-thaw treatment two times to destroy the cell membrane, the tissue homogenate was centrifuged at 5000 g (2-8°C) for 5 min to take the supernatant, which should be detected. For cells, centrifugation was performed at 1000 g (2-8°C) for 15 min, and the supernatant should be immediately detected.
Csb e09945h
Manufactured by Cusabio
Sourced in China
The CSB-E09945h is a laboratory equipment product manufactured by Cusabio. It is designed to perform a specific function, but a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation.
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4 protocols using csb e09945h
1
ELISA for Inflammatory Markers in Feces and Tissues
2
Histological Analysis of Organ Tissues
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Before histology analysis, tissues were immersed in neutral formaldehyde for at least 24 h. Frozen liver tissues were stained with oil red and the pathological scores were graded based on the number and size of stained fat droplets. Other organs which included kidney, intestine, pulmonary, heart and spleen were dehydrated and embedded in paraffin for Hematoxylin-eosin (HE) staining. Afterwards, oil red o staining was conducted with a microtome to acquire a 5 μm serial sections. Sections for immunofluorescence staining were then incubated with ZO-1 or occludin-3 antibody at 4 °C overnights. Then, the sections were washed with PBS for three times and treated with secondary antibody (dissolved in 1% BSA) for 1 h at 25 °C. Images were recorded with an Olympus BX43 fluorescence microscope. To analyse the content of LPS in tissue, intestines were taken and measured with an LPS ELISA kit (Cusabio, CSB-E09945h) following the manufacturer's instructions. Briefly, tissues were pre-treated with 1 × PBS. Reagents were added to samples and incubated for 4.5 h at 37 °C, and the absorbance was read at 540 nm. Standard curve was conducted using known amounts of LPS.
3
Measurement of LPS Levels in Carotid Plaques
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Lipopolysaccharide (LPS) levels in serum and in homogenates of carotid plaques were measured using a commercial ELISA kit (Product No. CSB-E09945h; Cusabio, Wuhan, China). Standards of LPS, purified from Escherichia coli, and samples were plated for 2 hours at room temperature onto a microplate pre-coated with the antibody specific for LPS. After incubation, samples were read at 450 nm. Values were expressed as pg/ml; intra-assay and inter-assay coefficients of variation (CVs) were 8% and 10%, respectively.
4
Measuring Gut Permeability and Inflammatory Markers
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Human LPS-binding protein (LBP) levels were measured by ELISA following the manufacturer’s instructions (DY870-05, RnD Systems) in the sera of RA patients and healthy controls (HC) (diluted 1 in 1000 in PBS/1% BSA). The level of intestinal fatty acid binding protein (I-FABP) was also measured in the sera of the mentioned RA patients and HC (1:1) by ELISA according to manufacturer’s instructions (RAB0537, Sigma). LPS levels were also measured by ELISA in these samples, undiluted, according to manufacturer’s instructions (CSB-E09945h, Cusabio).
Murine LPS-binding protein levels were measured in mouse serum (diluted 1 in 1000) by ELISA following the manufacturer’s instructions (HK205-01, Hycult Biotech).
For FITC-dextran translocation measurements, food was removed from cages for 14 h and mice were then gavaged with 600 mg/kg body weight of 4 kD FITC-dextran diluted in water (46944-500MG-F, Sigma). Blood was collected 30 min after gavage and the serum concentration of the FITC-dextran was determined using a fluorometer (TECAN Genios Spectra Fluor plus) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. FITC-dextran was serially diluted in PBS to establish a standard curve.
Murine LPS-binding protein levels were measured in mouse serum (diluted 1 in 1000) by ELISA following the manufacturer’s instructions (HK205-01, Hycult Biotech).
For FITC-dextran translocation measurements, food was removed from cages for 14 h and mice were then gavaged with 600 mg/kg body weight of 4 kD FITC-dextran diluted in water (46944-500MG-F, Sigma). Blood was collected 30 min after gavage and the serum concentration of the FITC-dextran was determined using a fluorometer (TECAN Genios Spectra Fluor plus) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. FITC-dextran was serially diluted in PBS to establish a standard curve.
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