Diaphragm muscles from at least 5 animals/strain (wild type, mdx, and moAb-Il6r-treated and untreated mdx mice) were homogenized in modified lysis buffer (Tris–HCl, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV4/0.2 mM, cocktail protease inhibitors/1 × (Sigma) and processed for western blot analysis. Filters were blotted with antibodies against: pStat3 (Tyr705) (cat #9145, Cell Signaling), Stat3 (cat # 9132, Cell Signaling); NFkBp65 (ser536) (cat # 3033, Cell Signaling); and NFkB (cat #4764, Cell Signaling). Signals were captured by ChemiDoc-It® Imaging System (UVP, LLC) and densitometric analysis was performed with VisionWorks®LS Image Acquisition and Analysis Software (UVP, LLC). ELISA assay was performed using either a human (to detect IL6; cat # HS600B) or mouse (to detect Il6, cat # M6000B, and Tnfα, cat # MTA00B) Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to manufacturer's protocol. Il2 expression was evaluated on the diaphragm of at least 3 animals/strain and with 2 biological replicates, using a RayBio® Mouse Antibody Array-G series (RayBiotech, Inc.), according to manufacturer's protocol. The intensities of signals were quantified with RayBio® Antibody Array Analysis Tool software.
Quantikine colorimetric sandwich elisa
Manufactured by R&D Systems
Sourced in United States
Quantikine® Colorimetric Sandwich ELISAs are a quantitative sandwich immunoassay designed to measure specific proteins in cell culture supernates, serum, plasma, urine and other biological fluids. The assay employs the quantitative sandwich enzyme immunoassay technique.
Automatically generated - may contain errors
Lab products found in correlation
Nf kb, by Cell Signaling Technology (2 mentions)
Visionworks ls image acquisition and analysis software, by Analytik Jena (2 mentions)
Chemidoc it imaging system, by Analytik Jena (2 mentions)
Cocktail protease inhibitors 1x, by Merck Group (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Nfkbp65 ser536, by Cell Signaling Technology (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Nitrotyrosine, by Merck Group (1 mentions)
Gp91phox, by BD (1 mentions)
Chemi doc xrs 2015, by Bio-Rad (1 mentions)
Hrp conjugated secondary antibody, by Fortis Life Sciences (1 mentions)
Criterion tgx stain free precast gel, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Cobas e411 analyzer, by Roche (1 mentions)
Vegf165, by R&D Systems (1 mentions)
Elx808 absorbance reader, by Agilent Technologies (1 mentions)
Bone scraper, by Hu-Friedy (1 mentions)
10 protocols using quantikine colorimetric sandwich elisa
1
Diaphragm Molecular Profiling in Dystrophy
2
Quantitative VEGF and CXCL8 Secretion
3
BAFF Levels in Mouse Sera
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Sera from 2 and 5 month old female mice were diluted in assay diluent at two-fold serial dilutions starting at 1:5 to 1:500. Sera BAFF levels were measured using Quantikine® Colorimetric Sandwich ELISAs (R&D Systems) according to the manufacturer’s protocol. BAFF concentrations in triplicate serum samples were determined by serial dilution of BAFF standard provided by kit.
4
Analyzing Dystrophic Diaphragm Muscle Signaling
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Diaphragm muscles from at least five animals/strain (wild-type, mdx and mdx/IL6 mice) were homogenized in modified lysis buffer [Tris–HCl, ph 7.5/20 mm , EDTA/2 mm , EGTA/2 mm , sucrose/250 mm , DTT/5 mm , Triton-X/0.1%, PMSF/1 mm , NaF/10 mm , SOV4/0.2 mm , cocktail protease inhibitors/1X (Sigma)]. Filters were blotted with antibodies against: Pax-7, Desmin, NFkBp65 (ser536), NFkB (Cell Signaling); GAPDH (Santa Cruz). Signals were captured by ChemiDoc-It® Imaging System (UVP, LLC) and densitometric analysis was performed with VisionWorks®LS Image Acquisition and Analysis Software. Elisa assay was performed to detect murine and transgenic IL-6 using Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to manufacturer's protocol.
5
Quantification of Eicosanoid Lipid Mediators
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The concentrations of TXB2, 5-HETE, 12-HETE, 15-HETE were determined by an ELISA (Quantikine® Colorimetric Sandwich ELISAs, R&D Systems, USA, Quantikine® Colorimetric Sandwich ELISAs, My BioSource, USA; Quantikine® Colorimetric Sandwich ELISAs, Cayman Chemical Company, USA).
6
Quantifying Oxidative Stress in Diaphragm Muscle
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Diaphragm muscles were isolated from 24-week-old wild-type and NSE/IL-6 mice, and liquid nitrogen powdered samples were homogenized in protein lysis buffer (Tris-HCl, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, Sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV4/0.2 mM, Cocktail Protease Inhibitors/1x (Sigma-Aldrich)). For western blotting analysis, 70 μg of protein extract was used, and filters were blotted with primary antibodies against gp91phox (BD Transduction), G6PD (Santa Cruz), Nitrotyrosine (Millipore), and GAPDH (Santa Cruz) and appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories). Nitrotyrosine quantification was performed using the Stain-Free blot method for normalization (Criterion TGX Stain-Free Precast Gels; Bio-Rad).
Signals were captured by Chemi Doc™ XRS 2015 (Bio-Rad Laboratories), and densitometric analysis was performed using Image Lab software (version 5.2.1; Bio-Rad Laboratories©).
For the evaluation of circulating human IL-6, an ELISA was performed on serum samples using Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to the manufacturer's protocol.
Signals were captured by Chemi Doc™ XRS 2015 (Bio-Rad Laboratories), and densitometric analysis was performed using Image Lab software (version 5.2.1; Bio-Rad Laboratories©).
For the evaluation of circulating human IL-6, an ELISA was performed on serum samples using Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to the manufacturer's protocol.
7
Cardiovascular Biomarker Profiling by Elecsys and Olink
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PlGF and sFLT1 concentrations were measured using Elecsys electrochemiluminescence immunoassays on a Cobas e411 analyzer (Roche Diagnostics). The NPX of 90 additional proteins associated with cardiovascular disease was measured using the Olink Cardiovascular II proximity extension assay (full list of proteins in Supplemental Table 17 ). In the discovery set, but not the validation set, VEGFA, VEGFD, VEGFR2, neuropilin 1 (NRP1), and endoglin were measured in triplicate using Quantikine colorimetric sandwich ELISAs (R&D Systems).
8
Functionalized MWNT for VEGF Delivery
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The versatile features of MWNT are functionalized, such as the multipurpose innovative carriers for drug delivery and diagnostic applications via physicochemical treatment. In our study, it means the MWNT are purified and treated by plasma polymerization to obtain the ability to carry and release VEGF165. Raw MWNT (Wako Pure Chemical Industries, Ltd, Osaka, Japan) was purchased and purified as previously reported.18 (link) VEGF165 was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Recombinant human VEGF165 was loaded into purified MWNT at a ratio of 1:10,000 (0.1 μg:1 mg) by solution sonication for 30 minutes. The suspensions were then freeze-dried for 48 hours. The VEGF165-loaded MWNT were coated with PLGA film by plasma polymerization to prepare the VEGF165-releasing device, as reported previously.19 The ratio of lactic acid to glycolic acid was 1:2. After plasma polymerization for 12 hours, the novel MWNT were characterized using high-resolution transmission electron microscopy (HRTEM). The amount of VEGF165 loaded into MWNT was analyzed by human VEGF enzyme-linked immunosorbent assay (ELISA) kits (Quantikine® Colorimetric Sandwich ELISAs, R&D Systems), according to the manufacturer’s instructions.
9
Porcine Bone Chip Media Protocols
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BCM was prepared as described.12 In brief, cortical bone chips were harvested from the buccal side of fresh pig mandibles (slaughterhouse: Küng Metzgerei, Toffen, Switzerland) using a bone scraper (Hu-Friedy, Rotterdam, The Netherlands) and were placed into either Ringer’s solution (RS) or Ringer’s solution mixed with autologous serum (RS+S) at a 1:1 ratio. A ratio of 5 g bone chips per 10 mL medium was used. Each of the two types of media, abbreviated as BCM-RS and BCM-RS+S, was supplemented with antibiotics and antimycotics. BCM from four independent preparations was collected at 10, 20, and 40 min and at 1, 3, and 6 days, sterile-filtered, and kept frozen at −80 °C.
The release of TGF-β1 or BMP-2 protein in BCM preparations was quantified using Quantikine® colorimetric sandwich ELISA (R&D Systems, Zug, Switzerland), according to the manufacturer’s procedure. Absorbance was measured at 450 and 570 nm using an ELx808 Absorbance Reader (BioTek, Luzern, Switzerland). Data represent mean±SD from three independent experiments performed in duplicate.
The release of TGF-β1 or BMP-2 protein in BCM preparations was quantified using Quantikine® colorimetric sandwich ELISA (R&D Systems, Zug, Switzerland), according to the manufacturer’s procedure. Absorbance was measured at 450 and 570 nm using an ELx808 Absorbance Reader (BioTek, Luzern, Switzerland). Data represent mean±SD from three independent experiments performed in duplicate.
10
Plasma Biomarker Quantification by ELISA
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Peripheral blood was collected into Acid Citrate Dextran (solution A) vacutainer tubes (BD Biosciences, Franklin Lakes, NJ USA) before being processed, with plasma stored directly at −80°C. Enzyme-linked immunosorbent assay (ELISA) kits were used to quantify plasma-soluble analytes in duplicate according to the manufacturers' instructions. Plasma concentrations of soluble vascular cell adhesion molecule (VCAM), C-reactive protein, and soluble CD14 were assayed by Quantikine® Colorimetric Sandwich ELISA (R&D systems, Minneapolis, MN USA). Plasma concentration of D-dimer was assayed using IMUCLONE D-dimer ELISA (Sekisui Diagnostics, Stamford, CT USA). V-PLEX Human Proinflammatory Panel II (4-Plex; Meso Scale Discovery, Gaithersburg, Maryland, USA) was used to quantify interleukin (IL)-6, interferon (IFN)-γ and tumor necrosis factor (TNF), according to manufacturer's instructions. Samples were re-assayed if intra-assay coefficient of variability between duplicates was >20%CV and sample absorbance reading was outside the assay standard range.
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