To evaluate the interaction among bacteria, differentiated epithelial H4-1 cells and macrophages, two different experimental approaches were conducted: 1) direct stimulation of MDBMs or TLT cells with bacteria (Fig. 1A andB) and 2) indirect stimulation of TLT cells via bacteria-challenged H4-1 cells seeded on Transwell filter inserts (gut model) (Fig. 1C). The apical surface of MDBMs, TLT and H4-1 monolayers was challenged by the addition of 106 CFU/ml of L. plantarum LS/07, 106 CFU/ml of LGG and 10 ng/ml LPS (from Escherichia coli 0111:B4, Sigma-Aldrich) respectively. The concentration 106 CFU/ml of bacteria was chosen based on previously performed cytotoxicity assay with crystal violet (data not shown). After 24 h co-cultivation at 37 °C in an atmosphere of air with 5% CO2, culture supernatants (from the basolateral side in the case of gut model) were harvested and stored at -20 °C until cytokine analysis. Cytokines were measured by commercially available ELISA kits for human IL-1b, IL-10, TNF-a and TGF-b1 (USCN Life Science Inc., USA), according to the manufacturer's instructions.
Il 10
Manufactured by USCN
Sourced in China
IL-10 is a cytokine that plays a crucial role in the regulation of the immune system. It is a pleiotropic cytokine that can inhibit the synthesis of a number of pro-inflammatory cytokines, as well as the expression of high-affinity Fc receptors on macrophages. IL-10 is primarily produced by monocytes, macrophages, and Th2 cells.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by USCN (1 mentions)
Oxiselect nitrotyrosine elisa kit, by Cell Biolabs (1 mentions)
Oxiselect tbars assay kit, by Cell Biolabs (1 mentions)
Polytron homogenizer pt 1200 e, by Kinematica (1 mentions)
Interleukin 6 (il 6), by USCN (1 mentions)
Il 1β, by USCN (1 mentions)
Interleukin 6 (il 6), by Cusabio (1 mentions)
Tnf α, by USCN (1 mentions)
4 protocols using il 10
1
Investigating Bacterial-Epithelial-Macrophage Interactions
2
Cytokine Profiling in Brain Tissue
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The frozen brains were homogenized in 100 mg tissue/mL cold PBS. The samples were centrifuged at 12,000 × g for 15 min. The supernatant was collected for a protein assay using a BCA protein assay reagent kit (PIERCE, Milwaukee, WI). The serum and brain levels of IL-1β (eBioscience, Vienna, Austria), TNF-α (eBioscience Vienna, Austria), PGE2 (R&D Systems, Minneapolis, MN), IL-4 (USCN, Wuhan, China), and IL-10 (USCN, Wuhan, China) were measured using ELISA kits according to the manufacturer’s instructions.
3
Lung Cytokine and Oxidative Stress Assay
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Concentrations of cytokines and oxidative modification products were determined in 10% (weight/volume) lung homogenate prepared using 0.1 M ice-cold phosphate buffer (PBS, pH 7.4) by Polytron homogenizer PT 1200 E (5-times for 25 s, 1200 rpm; Kinematica AG, Switzerland). Homogenates were then frozen 3 times and centrifuged (12,000 rpm, 15 min, 4 °C). Final supernatants were then stored at −70 °C until the analysis was performed.
Concentrations of IL-6, IL-1β, IL-10 (USCN Life Science Inc., Wuhan, China), and RAGE (MyBioSource, San Diego, CA, USA) were quantified using rabbit-specific ELISA kits according to the manufacturers’ instructions. Data were expressed in pg/mL.
Protein oxidative damage was determined using OxiSelectTM Nitrotyrosine ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed in 3-nitrotyrosine nanomole concentration (nM, 3NT). Lipid oxidative damage expressed by the concentration of thiobarbituric acid reacting substances (TBARS) was determined by OxiSelectTM TBARS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed as malondialdehyde in micromole concentration (μM MDA).
Total antioxidant capacity (TAC) was determined using an ELISA kit according to the manufacturer’s instructions (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed as micromole concentration of copper reducing equivalents (μM CRE).
Concentrations of IL-6, IL-1β, IL-10 (USCN Life Science Inc., Wuhan, China), and RAGE (MyBioSource, San Diego, CA, USA) were quantified using rabbit-specific ELISA kits according to the manufacturers’ instructions. Data were expressed in pg/mL.
Protein oxidative damage was determined using OxiSelectTM Nitrotyrosine ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed in 3-nitrotyrosine nanomole concentration (nM, 3NT). Lipid oxidative damage expressed by the concentration of thiobarbituric acid reacting substances (TBARS) was determined by OxiSelectTM TBARS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed as malondialdehyde in micromole concentration (μM MDA).
Total antioxidant capacity (TAC) was determined using an ELISA kit according to the manufacturer’s instructions (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed as micromole concentration of copper reducing equivalents (μM CRE).
4
Measuring Inflammatory and Oxidative Stress Markers in Rats
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ELISA kits were used to detect the inflammatory factors interleukin (IL)-1β (cat. no. CSB-E08055r; CUSABIO Technology), IL-6 (cat. no. SEA079Ra; Wuhan USCN Business Co., Ltd.), TNF-α (cat. no. SEA133Si; Wuhan USCN Business Co., Ltd.) and IL-10 (cat. no. SEA056Ra; Wuhan USCN Business Co., Ltd.) in the serum from the rats. In addition, the oxidative stress indicators superoxidase dismutase (SOD; cat. no. SES134Hu), malondialdehyde (MDA; cat. no. CEA597Ge), nitric oxide (NO; cat. no. IS100) and the intestinal injury markers D-lactic acid in serum (cat. no. CEV643Ge), diamine oxidase (DAO; cat. no. SEJ298Hu) and intestinal fatty acid-binding protein (I-FABP; cat. no. SEA559Ra) were detected in the serum using kits from Wuhan USCN Business Co., Ltd. The tests were performed according to the manufacturer's instructions.
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