BAL fluid was analyzed using a cytokine antibody microarray from RayBiotech Inc. (Norcross GA). Pooled samples from N = 6 MDI skin sensitized mice, exposed to either GSH–MDI or MDI control (MDI reacted without GSH = MDI-m as described above), were incubated on different array membranes overnight at 4 °C. Microarrays were developed according to the manufactures specifications using enhanced chemiluminescence with substrate from Thermo Fisher Scientific Inc. and Carestream Kodak BioMax light film from Sigma-Aldrich. Additional cytokine analyses were performed by ELISA using kits from Biolegend (IL-4, IL-5, IL-10, IL-12p40, IL-23), R&D Diagnostics (IL-13, IL-12p70), or eBiosciences (San Diego, CA) (IL-10) and RayBiotech Inc. (IL-10) according to the manufacturers' recommendations.
Il 10
Manufactured by RayBiotech
Sourced in United States, Canada, China
IL-10 is a cytokine that plays a key role in regulating the immune system. It is produced by a variety of cell types, including T cells, B cells, and monocytes. IL-10 has anti-inflammatory properties and helps to suppress the immune response.
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Lab products found in correlation
Interleukin 6 (il 6), by RayBiotech (6 mentions)
Tnf α, by RayBiotech (6 mentions)
Il 1β, by RayBiotech (5 mentions)
Mcp 1, by RayBiotech (4 mentions)
Interleukin 4 (il 4), by RayBiotech (4 mentions)
Il 13, by RayBiotech (3 mentions)
Il 17, by Jiangsu Meimian (3 mentions)
Interleukin 2 (il 2), by RayBiotech (2 mentions)
Il 10, by R&D Systems (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Il 1ra, by R&D Systems (1 mentions)
Tgf β, by R&D Systems (1 mentions)
20 r rg3, by Yuanye Bio-Technology (1 mentions)
Cell cycle kit, by BD (1 mentions)
Cyclophosphamide cp, by Merck Group (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Il 18, by LifeSpan BioSciences (1 mentions)
Mcp 1, by Cusabio (1 mentions)
Il 17, by Cusabio (1 mentions)
Il 12, by Elabscience (1 mentions)
19 protocols using il 10
1
Cytokine Profiling of MDI-Sensitized Mice
2
Cytokine Secretion Quantification
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The cells with a density of 1 × 105 cells per well were seeded on samples on 24-well plates for 4 d. The cell culture medium was collected to detect the concentration of interleukin-1 receptor antagonist (IL-1ra; R&D System, USA), IL-4 (Anogen, Canada), IL-6 (Anogen, Canada), IL-10 (Raybiotech, USA), transforming growth factor-β (TGF-β; R&D System, USA) and tumor necrosis factor-α (TNF-α; Anogen, Canada) by ELISA. A microplate reader (Thermo Fisher Scientific Inc., USA) was used to detect the absorbance of the plate according to the protocol. The concentrations of the above-mentioned factors were calculated using standard curves.
3
Quantification of Ginsenosides and Monosaccharides
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The reference substances of ginsenosides (nR1, Rg1, Re, pF11, Rf, Ra2, Rb1, Rc, Ro, Rb2, Rb3, Rd and 20(R)-Rg3) and monosaccharides (Glc, Ara, Gal, Man, GalA, Rha, Rib, ClcUA, Xyl and Fuc) were purchased from Shanghai Yuanye Biotechnology (Shanghai, China). The purity of these references was higher than 99.0% indicated by HPLC analysis. Cyclophosphamide (CP) was supplied by Sigma-Aldrich Company Ltd., (United States). Thrombopoietin (TPO), erythropoietin (EPO), and granulocyte-macrophage colony stimulating factor (GM-CSF) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Quantibody®Array Glass Chip of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, IFNγ, TNF-α and MCP-1 were purchased from RayBiotech Life, Inc. (United States). FITC-Annexin-V and Cell Cycle kits were purchased from BD Biosciences Pharmingen (United States). The blood cell analysis reagent kit was purchased from IDEXX Laboratories Inc (United States). Protopanaxadiol saponins (PDS) and protopanaxatriol saponins (PTS) were supplied by Professor Chen Yanping in the College of Chemistry, Jilin University; PDS and PTS were LC-MS grade. The voucher specimen of PDS (S20190011) and PTS (S20190012) were deposited in the authors’ lab in Changchun University of Chinese Medicine (Changchun, China).
4
Enzyme-Linked Immunosorbent Assays for Renal Biomarkers
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Commercial ELISA kits were utilized for the determination of renal myeloperoxidase (MPO; Hycult Biotech, Uden, Netherlands), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10 (RayBiotech, USA), MCP-1 (Cusabio Biotech) and IL-18 (LifeSpan Biosciences, USA), as instructed by the manufacturer.
5
Cytokine Profiling in Serum Samples
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Recipient serum was obtained from peripheral blood 10 days before surgery and when euthanasia was performed or death observed. Cytokine concentrations were measured by ELISA kits as described by the manufacturers, including comparisons with standard curves. Kits used and minimum detectable concentrations of interleukins (IL): IL-4: 1.5 pg/ml (RayBiotech Inc., Norcross, GA, USA); IL-10: 10 pg/ml (RayBiotech Inc., Norcross, GA, USA); IL-12: 9.375 (Elabscience, Bethesda, MD, USA); IL-17: 23.43 pg/ml (Cusabio, Hubei, China); IL-21: 3.3 pg/ml (Merck-Millipore, Billerica, MA, USA); IL-23: 0.196 pg/ml (Merck-Millipore, Billerica, MA, USA); tumor necrosis factor-alpha (TNF-a): 15 pm/ml (Diaclone, Besancon Cedex, France); and transforming growth factor (TGF)-b1: 11.4 pg/ml (Milliplex ® , Merck-Millipore, Billerica, MA, USA).
6
Multiplex Analysis of Rat Synovial Inflammation
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Rats were euthanized via CO2 inhalation at three days or twenty-one days post-surgery for both the naïve group and the MMT surgery group. Synovial fluid was collected by first injecting 100 μl of saline intra-articularly using a 30 gauge insulin syringe, followed by aspirating approximately 50 to 100 μl of the synovial fluid and saline using the same syringe. Synovial fluid was analyzed using the Quantibody® Rat Inflammation Array 1, a multiplex ELISA kit that quantitatively measured 10 rat inflammatory factors: IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, monocyte chemoattractant protein-1 (MCP-1), and TNFα (RayBiotech, Norcross, GA, USA).
7
Inflammasome Activation in Immune Cells
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LPS and pancreatic enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA); lymphocyte separation medium was purchased from Jingyang Biological (Tianjin, China); fetal bovine serum (FBS) was obtained from GIBCO (Australia); RPMI-1640 glucose free medium was purchased from Hyclone (Logan, UT, USA); RNeasy Mini Kit, RNase-Free DNase Set, RT2 First Strand Kit, RT2 SYBR® Green ROX qPCR Mastermix, and Human Inflammasomes PCR Array were obtained from Qiagen (Valencia, CA, USA). ELISA kits of human TNF-α, IL-1β, IL-6, IL-10, and MCP-1 were purchased from RayBiotech (Atlanta, USA). Anti-NLRP3 and anti-Caspase-1 antibodies were purchased from Abcam (Cambridge, United Kingdom). Secondary antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA).
8
Cytokine Secretion of Macrophages
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Macrophages with a density of 1 × 105 cells per well were seeded on the samples (three replicates) for 4 days. The cell culture medium was collected, centrifuged at 1500 rpm at 4°C to get the supernatant, and stored in the sterile 1.5 ml tube for use. The supernatant of culture medium was used to measure the concentration of interleukin-4 (IL-4; Anogen, Canada), IL-6 (Anogen, Canada), IL-10 (Raybiotech, USA) and TNF-α (Anogen, Canada) by enzyme-linked immunosorbent assay (ELISA). The absorbance of the plate was detected by a microplate reader according to the protocol. The concentrations of the cytokines were calculated by using the corresponding standard curves.
9
Alveolar Bone Loss Analysis in Mice
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The maxillae of each mouse collected at the endpoint were defleshed and soaked in toluidine blue solution. Stained maxillae were photographed using a dissection microscope (Nikon), and total alveolar bone loss was calculated by measuring the cemento-enamel junction (CEJ) to the alveolar bone crest decalcification (ABC) distances on the buccal side of each root [41 (link)]. Some maxillae were fixed in 10% formaldehyde and then 10% EDTA solution at 4 °C for 3 to 4 weeks. The decalcified maxillae samples were then embedded in paraffin and sectioned (7-μm thickness) for TRAP and hematoxylin and eosin (H&E) staining. For immunofluorescence staining, the decalcified maxillae samples were embedded in OCT compound (Fisher Scientific) and sectioned (6 μm thickness) using a cryostat. The expression of Sema4D was monitored using biotin-conjugated anti-Sema4D-mAb, followed by avidin conjugated to AlexaFluor 488. Gingival tissue homogenates were obtained from the gingival tissue of each mouse at the endpoint, and Sema4D, TNF-α, RANKL, OPG, IL-10 and IGF-1 production in the homogenate was analyzed using an ELISA kit purchased from the following sources: Sema4D: RayBiotech; TNF-α, RANKL, OPG, IL-10 and IGF-1: R&D Systems.
10
Quantifying Neuroimmune Factors by ELISA
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Enzyme-linked immunosorbent assay (ELISA) kits were used to quantify levels of netrin-1 (MEIMIAN, China), IL-17 (MEIMIAN, China) and IL-10 (RayBiotech, United States) in duplicate wells containing serum and CSF samples according to the manufacturers’ protocols. Blood was collected via a right ventricular puncture and about 1–2 ml serum per rat was obtained following centrifugation at 3000 rpm for 5 min. Approximately 100 μl of CSF was collected from each rat via foramen magnum puncture. Briefly, for detection of IL-10, samples were incubated for 2.5 h in a microtiter plate pre-coated with monoclonal antibody, and then for 60 min with biotinylated antibody at room temperature, followed by the addition of streptavidin solution for 45 min and one-step substrate reagent for 30 min as performed in the dark at 37°C. For netrin-1 and IL-17, each sample and its control were incubated in the 96-well microtiter plate for 0.5 h at 37°C, followed by HRP-conjugated reagent and chromogen solution at 37°C. All optical densities were measured at 450 nm. Each sample was detected in duplicated wells and the results were averaged.
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