Conditioned supernatants were taken from the cell culture medium and stored at −80°C for subsequent examination. Secreted HA, IL-6, pentraxin 3 (PTX3), and cyclooxygenase-2 (COX-2) amounts were detected with commercial ELISA kits (HA, R&D Systems, Minneapolis, MN, USA; IL-6, PTX3 and COX-2, Elabscience Biotechnology, Wuhan, China), as directed by the respective manufacturers. Triplicate assays were carried out.
Cox 2
Manufactured by Elabscience
Sourced in United States
COX-2 is a laboratory equipment used for the detection and quantification of the cyclooxygenase-2 enzyme. It is a crucial component in the study of inflammatory processes and the development of anti-inflammatory drugs.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by Elabscience (2 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Chemiluminescence western blot detection system, by Bio-Rad (1 mentions)
β actin, by Elabscience (1 mentions)
Lps from escherichia coli serotype 0111 b4, by Merck Group (1 mentions)
Il 1β, by Elabscience (1 mentions)
Il 10, by Elabscience (1 mentions)
4 protocols using cox 2
1
Quantification of Secreted Factors
2
Immunomodulatory Effects of hUC-MSCs
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hUC-MSCs were treated with different combinations of IL-1β, TNF-α and IFN-γ for 24 hours. The cell supernatants were centrifuged and collected and then analyzed by IL-6 (R&D system, USA), IL-8 (Elabscience, China), CCL2 (Elabscience, China), COX-2 (Elabscience, China), TSG-6 (SenBeiJia, China) and IDO1 ELISA kit (SenBeiJia, China) ELISA kits according to the manufacturer’s protocols.
3
Liver Protein Extraction and Western Blot
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The collected liver samples were washed twice with cold phosphate-buffered saline at 800×g for 10 min. Total protein was extracted using a radio-immunoprecipitation assay buffer and the protein concentration was measured by the BCA method. The proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Shanghai Jinsheng Biological Engineering Co., Ltd). The membranes were blocked with 50% bovine serum albumin containing 0.05% Tween-20 in Tris buffered-saline for 4 h at room temperature. After 4 h, the polyvinylidene difluoride membrane was incubated with the primary antibody for the whole night. The membranes were then washed three times (5 min each), and incubated with secondary antibodies for 45 min at room temperature. The protein bands for COX-2, iNOS, and β-actin (Elabscience Biotechnology Co., Ltd) were identified with a chemiluminescence western blot detection system (Bio-Rad, Hercules, CA, USA).
4
Flavonoids Modulate Inflammatory Responses
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The cells were seeded on the well bottom at a density of 1 × 105 cells/mL and incubated for 24 h. To induce the production of inflammatory factors, the cells were stimulated with lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Sigma) (10 μg/mL for 2 h). Diosmin or diosmetin were added to the cell culture 2 h before LPS stimulation and then incubated for 24 h with LPS or 24 h after LPS stimulation and then incubated for another 2 h. After that time, culture supernatants were collected and analyzed. The levels of IL-1β, IL-6, IL-10, COX-2, and PGE2 (Elabscience, Houston, TX, USA) were measured immunoenzymatically (ELISA) using commercially available kits according to the manufacturer’s instruction. The absorbance was measured using a microplate reader at 450 nm. A stock solution of the flavonoid was prepared using DMSO/culture medium (1:1) and it was diluted. The final concentration of DMSO did not exceed 0.5% and this concentration did not affect the cell viability.
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