Culture supernatants collected after S-layer stimulation of BMDCs or the BMDC/T cell co-culture were analyzed for the pro-inflammatory cytokines IL-6 and TNF (DuoSet ELISAkits, R&D Systems, Minneapolis, MN, USA), the anti-inflammatory cytokines IL-10 (ABTS ELISA Development Kit, PeproTech, Hamburg, Germany) and TFG-β (DuoSet ELISAkits, R&D Systems, Minneapolis, MN, USA) and the T cell secreted cytokines IL-17 and IFN-γ (ABTS ELISA Development Kit, PeproTech, Hamburg, Germany) according to the manufacturer's instructions. Plates were developed with the substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) and the color reaction was stopped with 2 M sulfuric acid. Absorbance was measured at 450 nm with a wavelength correction at 570 nm using a MultiskanGo microplate spectrophotometer.
Abts elisa development kit
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, Germany
The ABTS ELISA Development kits are a set of reagents designed for the development of enzyme-linked immunosorbent assay (ELISA) protocols. The core function of these kits is to provide the necessary components, including the ABTS substrate, to enable the colorimetric detection and quantification of target analytes in various sample types.
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Lab products found in correlation
Cobas 8000, by Roche (3 mentions)
Human pon1 elisa kit, by Elabscience (2 mentions)
Human mouse bdnf duoset elisa, by R&D Systems (2 mentions)
Gdnf elisa kit, by Abcam (2 mentions)
Substrate reagent, by R&D Systems (2 mentions)
Duoset elisa kit, by R&D Systems (2 mentions)
Varioskan, by Thermo Fisher Scientific (1 mentions)
Dy999, by R&D Systems (1 mentions)
Elisa kit, by Elabscience (1 mentions)
Elisa kit, by Proteintech (1 mentions)
Duoset elisa development system, by R&D Systems (1 mentions)
Human galectin 3 quantikine elisa kit, by R&D Systems (1 mentions)
Angiotensin 2 eia kit, by Merck Group (1 mentions)
In nite f200 pro microplate reader, by Tecan (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Recombinant mouse ifn β, by PBL Assay Science (1 mentions)
B16 blue ifn α β reporter cells, by InvivoGen (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
Abts elisa buffer kit, by Thermo Fisher Scientific (1 mentions)
Human ll 37 elisa kit, by Hycult Biotech (1 mentions)
30 protocols using abts elisa development kit
1
Cytokine Quantification in BMDC Cultures
2
Quantifying bFGF and EGF Levels
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Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were determined by using human FGF-basic standard ABTS ELISA development kit and human EGF standard ABTS ELISA development kit (PeproTech Inc., Rocky Hill, NJ, USA), respectively. After treatment according to indicated protocol, the supernatant was collected and discarded cell debris by centrifugation. Then, the supernatant was incubated in pre-blocked capture antibody-coated 96-well plate for 2 h at room temperature (RT). Then, the detection antibody was added and incubated for 2 h at RT. After that, the avidin-HRP-conjugated anti IgG was added and incubated for 30 mins at RT. After adding a substrate, the luminescence was determined by using microplate reader at 450/605 nm.
3
Cytokine Quantification in Cell Culture
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Supernatant from cell culture was recovered at 1, 3 and 7 days after treatment and cytokines were measured according the human IL-6 and IL-8 Standard ABTS ELISA Development Kits (PeproTech). Optical density was measured at 405 nm with Varioskan (ThermoFisher).
4
Inflammatory Cytokine Quantification
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The plasma and tissue levels of the inflammatory cytokines (TNF-α, IL-6, and IL-1β) were measured using the enzyme-linked immunosorbent assay (ELISA) technique (Stat Fax 303 Plus Microstrip Reader, Minneapolis, MN, USA), with commercially available kits (rat TNF-α, IL-6, and IL-1β ABTS ELISA Development kits, PeproTech EC, Ltd., London, UK).
5
Inflammatory Cytokine Assessment in Aortic Tissue
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Fragments from the ascending aorta were taken, weighed, and homogenized in four volumes of phosphate-buffered saline solution. The homogenization was made using an automated Witeg Homogenizer (HG-15D, Wertheim, Germany) at 27,000 rpm. Right after the homogenate was centrifuged (15,000 rpm at 4 °C for 15 min), the clear supernatant was used for pro-inflammatory cytokine analysis. The tissue levels of TNF-α, IL-6, and IL-1β, were assessed using the ELISA technique (Stat Fax 303 Plus Microstrip Reader, Minneapolis, USA), with commercially available kits (rat TNF-α, IL-6, and IL-1β ABTS ELISA development kits, PeproTech EC, Ltd., London, UK).
6
Lipigenine Stimulates HBD-2 Production
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EXAMPLE 5
Lipigenine™ was tested for its ability to stimulate an increase in HBD-2 concentration. The HBD-2 standard ABTS ELISA development kits were obtained from PeproTech (Cat# 900-K172). ELISA was performed according to the manufactory instructions of each kit by adding 100 μl/well of culture medium after overnight treatment. The substrate of ELISA reaction was using the substrate reagent from R&D Systems (DY999), and the reactions were stopped by adding 50 μl of 1N H2SO4 in each well. The Lipigenine™ culture was compared to the control medium which contained no other ingredients. The results were measured using a colorimeter, absorbance was measured at 450 nanometers (nm) within 30 minutes. Wavelength correction was set to 570 nm. The concentration of each sample was calculated using ELISA standard curve. [000126] The addition of Lipigenine™ showed increased HBD-2 concentrations at both 0.1% and 1% Lipigenine™ in solution as compared to the control. An increase in HBD-2 concentration of 7% was observed for a 0.1% Lipigenine™ formulation while an increase in HBD-2 expression of 23% was observed for a 1% Lipigenine™ formulation. These results are shown in
7
Biomarkers of Inflammation and Stress
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Proinflammatory cytokines TNF-a, IL-1b, IL6, and cortisol were measured using the ELISA technique (Stat Fax 303 Plus Microstrip Reader, Palm City, FL, USA). Proinflammatory cytokines detection and quantification were performed using commercially ABTS ELISA development kits (PeproTech EC, Ltd., London, UK). Cortisol was determined using a rat COR ELISA kit; (FineTest, Wuhan Fine Biotech Co., Wuhan, China).
8
Lipigenine Stimulates HBD-2 Expression
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Example 5
Lipigenine™ was tested for its ability to stimulate an increase in HBD-2 concentration. The HBD-2 standard ABTS ELISA development kits were obtained from PeproTech (Cat #900-K172). ELISA was performed according to the manufactory instructions of each kit by adding 100 μl/well of culture medium after overnight treatment. The substrate of ELISA reaction was using the substrate reagent from R&D Systems (DY999), and the reactions were stopped by adding 50 μl of 1N H2SO4 in each well. The Lipigenine™ culture was compared to the control medium which contained no other ingredients. The results were measured using a colorimeter, absorbance was measured at 450 nanometers (nm) within 30 minutes. Wavelength correction was set to 570 nm. The concentration of each sample was calculated using ELISA standard curve.
The addition of Lipigenine™ showed increased HBD-2 concentrations at both 0.1% and 1% Lipigenine™ in solution as compared to the control. An increase in HBD-2 concentration of 7% was observed for a 0.1% Lipigenine™ formulation while an increase in HBD-2 expression of 23% was observed for a 1% Lipigenine™ formulation. These results are shown in
9
Measuring PON1 Activity and Cytokines
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Blood samples were obtained before, and one month after, RT administration. Samples for conventional biochemical and hematological analyses were processed immediately. Serum and EDTA-plasma samples for measurement of cytokine and PON1-related variables were stored at −80 °C until batched analyses. Serum PON1 activity was measured as the rate of hydrolysis of phenylacetate at 280 nm, in a 9 mM Tris-HCl buffer, pH 8.0, supplemented with 0.9 mM CaCl2, as previously reported [16 (link)]. PON1 can hydrolyze multiple substrates, but we chose phenylacetate because it is not toxic, the assay is simple, and is little influenced by PON1 gene polymorphisms [16 (link)]. Serum PON1 concentrations were determined using an in-house ELISA with rabbit polyclonal antibodies specific to PON1 [33 (link)]. PON1 specific activity was calculated as the ratio between the activity and the concentration, and is a measure of the activity per molecule. Plasma concentrations of CCL2, IL-4, and IFN-γ were measured by ABTS ELISA Development kits (Peprotech, London, UK). Standard biochemical and hematological analyses were performed in a COBAS® 8000 (Roche Diagnostics, Basel, Switzerland) and a Sysmex XN-1000TM (Sysmex GmbH, Norderstedt, Germany) analyzers, respectively.
10
Serum Biomarkers in Experimental Study
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The blood samples were taken from retroorbital plexus at the end of the experiment. One milliliter of blood on anticoagulants was preserved, and the serum was obtained through centrifugation within first hour. The obtained serum samples were kept at −80 °C until determinations were performed.
The serum levels of AST, ALT, and CK-Mb were determined through a spectrophotometric method using an automatic analyzer Applied Biosystem (Costa Brava, Barcelona (Spain)).
The serum levels of the inflammatory cytokines TNF-α, IL-6, and IL-1β were measured using the ELISA Stat Fax 303 Plus Microstrip Reader (Minneapolis, MN, USA) with commercially available kits (rat TNF-α, IL-6, and IL-1β ABTS ELISA Development kits, PeproTech EC, Ltd., London, UK). Troponin was also measured using an Elabscience ELISA kit. The determinations were done according to the manufacturer’s instructions. For each assay, samples were diluted as needed, and protein levels were calculated using a four-parameter logistic (4-PL) curve fit.
The serum levels of AST, ALT, and CK-Mb were determined through a spectrophotometric method using an automatic analyzer Applied Biosystem (Costa Brava, Barcelona (Spain)).
The serum levels of the inflammatory cytokines TNF-α, IL-6, and IL-1β were measured using the ELISA Stat Fax 303 Plus Microstrip Reader (Minneapolis, MN, USA) with commercially available kits (rat TNF-α, IL-6, and IL-1β ABTS ELISA Development kits, PeproTech EC, Ltd., London, UK). Troponin was also measured using an Elabscience ELISA kit. The determinations were done according to the manufacturer’s instructions. For each assay, samples were diluted as needed, and protein levels were calculated using a four-parameter logistic (4-PL) curve fit.
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