Mouse CX3CL1/Fractalkine DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant mouse CX3CL1/Fractalkine. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: CX3CL1/Fractalkine
CX3CL1/Fractalkine is a transmembrane adhesion protein with a chemokine domain separated from the membrane by a mucin stalk. It is upregulated on many cell types during inflammation. The chemokine and mucin regions can be shed as a soluble chemokine that signals through the receptor CX3CR1. During extravasation, membrane-bound CX3CL1 traps leukocytes, then is cleaved to allow diapedesis. Soluble CX3CL1 protects neurons from microglial neurotoxicity, recruits macrophages for wound healing, recruits osteoclast precursors for bone resorption, and contributes to pathogenesis in coronary artery disease.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Mouse CX3CL1/Fractalkine DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
11
Citations: Showing 1 - 10
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Chronic hyperpalatable diet induces impairment of hippocampal-dependent memories and alters glutamatergic and fractalkine axis signaling
Authors: Ribeiro, R;Silva, EG;Moreira, FC;Gomes, GF;Cussat, GR;Silva, BSR;da Silva, MCM;de Barros Fernandes, H;de Sena Oliveira, C;de Oliveira Guarnieri, L;Lopes, V;Ferreira, CN;de Faria, AMC;Maioli, TU;Ribeiro, FM;de Miranda, AS;Moraes, GSP;de Oliveira, ACP;Vieira, LB;
Scientific reports
Species: Mouse
Sample Types: Serum, Tissue Homogenates
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Inhibition of CSF1R, a receptor involved in microglia viability, alters behavioral and molecular changes induced by cocaine
Authors: MCM da Silva, GF Gomes, H de Barros, AM da Silva, AL Teixeira, FA Moreira, AS de Miranda, ACP de Oliveir
Scientific Reports, 2021-08-06;11(1):15989.
Species: Mouse
Sample Types: Tissue Homogenates
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Elevated circulating TGF?1 during acute liver failure activates TGF?R2 on cortical neurons and exacerbates neuroinflammation and hepatic encephalopathy in mice
Authors: M McMillin, S Grant, G Frampton, AD Petrescu, E Williams, B Jefferson, A Thomas, A Brahmarout, S DeMorrow
J Neuroinflammation, 2019-04-02;16(1):69.
Species: Mouse
Sample Types: Tissue Homogenates
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Demyelination induced by oxidative stress is regulated by sphingosine 1-phosphate receptors
Authors: SA O'Sullivan, M Velasco-Es, KK Dev
Glia, 2017-04-04;0(0):.
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PGI2 Controls Pulmonary NK Cells That Prevent Airway Sensitization to House Dust Mite Allergen
Authors: Bryan Simons
J. Immunol, 2016-11-28;0(0):.
Species: Mouse
Sample Types: BALF
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Glycogen synthase kinase-3? regulates fractalkine production by altering its trafficking from Golgi to plasma membrane: implications for Alzheimer's disease
Authors: Jesús Ávila
Cell. Mol. Life Sci., 2016-11-10;0(0):.
Species: Mouse
Sample Types: Tissue Homogenates
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NKG2D ligand overexpression in lupus nephritis correlates with increased NK cell activity and differentiation in kidneys but not in the periphery.
Authors: Spada R, Rojas J, Perez-Yague S, Mulens V, Cannata-Ortiz P, Bragado R, Barber D
J Leukoc Biol, 2015-01-12;97(3):583-98.
Species: Mouse
Sample Types: Cell Culture Supernates
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CX3CL1 expression in the conjunctiva is involved in immune cell trafficking during toxic ocular surface inflammation.
Authors: Denoyer A, Godefroy D, Celerier I, Frugier J, Riancho L, Baudouin F, Rostene W, Baudouin C
Mucosal Immunol, 2012-06-13;5(0):702.
Species: Mouse
Sample Types: Tissue Homogenates
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Chemokine binding protein M3 of murine gammaherpesvirus 68 modulates the host response to infection in a natural host.
Authors: Hughes DJ, Kipar A, Leeming GH, Bennett E, Howarth D, Cummerson JA, Papoula-Pereira R, Flanagan BF, Sample JT, Stewart JP
PLoS Pathog., 2011-03-17;7(3):e1001321.
Species: Mouse
Sample Types: Tissue Homogenates
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Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues.
Authors: Cardona AE, Sasse ME, Liu L, Cardona SM, Mizutani M, Savarin C, Hu T, Ransohoff RM
Blood, 2008-03-17;112(2):256-63.
Species: Mouse
Sample Types: Tissue Homogenates
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Elevated neuronal expression of CD200 protects Wlds mice from inflammation-mediated neurodegeneration.
Authors: Chitnis T, Imitola J, Wang Y, Elyaman W, Chawla P, Sharuk M, Raddassi K, Bronson RT, Khoury SJ
Am. J. Pathol., 2007-05-01;170(5):1695-712.
Species: Mouse
Sample Types: Tissue Homogenates
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