Mouse CXCL4/PF4 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 CXCL4/PF4. 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
Block Buffer: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Reagent Diluent: 0.1% BSA, 0.05% Tween 20 in Tris-buffered Saline (20 mM Trizma base, 150 mM NaCI) 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: CXCL4/PF4
CXCL4/PF4 is a secreted chemokine that is expressed by megakaryocytes and restrains the coagulation cascade. It is released by activated platelets and regulates Thrombin/Thrombomodulin complexes, activated Protein C (APC), Coagulation Factor Xa, and Fibrin. The anticoagulant heparin neutralizes CXCL4 procoagulant effects. Complexes of heparin and CXCL4 trigger antibody formation that causes the pathological syndrome HITT. Immunogenic complexes of CXCL4 with Apolipoprotein H contribute to antiphospholipid syndrome (APS). CXCL4 interferes with the proliferative and angiogenic functions of FGF-2 and VEGF. However, it can also exert proinflammatory and pro-atherogenic effects on monocytes, macrophages, and endothelial cells. CXCL4 may signal through CXCR3B in humans.
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 of Block Buffer 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 CXCL4/PF4 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.
13
Citations: Showing 1 - 10
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Genetic platelet depletion is superior in platelet transfusion compared to current models
Authors: M Salzmann, WC Schrottmai, JB Kral-Point, M Mussbacher, J Volz, B Hoesel, B Moser, S Bleichert, S Morava, B Nieswandt, JA Schmid, A Assinger
Haematologica, 2019-09-19;0(0):.
Species: Mouse
Sample Types: Plasma
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Evidence of CD40L/CD40 pathway involvement in experimental transfusion-related acute lung injury
Authors: S Tariket, H Hamzeh-Cog, S Laradi, CA Arthaud, MA Eyraud, T Bourlet, P Berthelot, O Garraud, F Cognasse
Sci Rep, 2019-08-29;9(1):12536.
Species: Mouse
Sample Types: Plasma
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Inhibition of profibrotic microRNA-21 affects platelets and their releasate
Authors: T Barwari, S Eminaga, U Mayr, R Lu, PC Armstrong, MV Chan, M Sahraei, M Fernández-, T Moreau, J Barallobre, M Lynch, X Yin, C Schulte, F Baig, R Pechlaner, SR Langley, A Zampetaki, P Santer, M Weger, R Plasenzott, M Schosserer, J Grillari, S Kiechl, J Willeit, AM Shah, C Ghevaert, TD Warner, C Fernández-, Y Suárez, M Mayr
JCI Insight, 2018-11-02;3(21):.
Species: Mouse
Sample Types: Plasma
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Chronic Intake of the Selective Serotonin Reuptake Inhibitor Fluoxetine Enhances Atherosclerosis
Authors: M Rami, R Guillamat-, P Rinne, M Salvermose, L Ring, M Bianchini, X Blanchet, RTA Megens, Y Döring, B Walzog, O Soehnlein, C Weber, A Faussner, S Steffens
Arterioscler. Thromb. Vasc. Biol., 2018-03-22;0(0):.
Species: Mouse
Sample Types: Serum
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Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor
Authors: C Daly, X Qian, C Castanaro, E Pasnikowsk, X Jiang, BR Thomson, SE Quaggin, N Papadopoul, Y Wei, JS Rudge, G Thurston, GD Yancopoulo, S Davis
Sci Rep, 2018-01-11;8(1):505.
Species: Mouse
Sample Types: Plasma
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Overexpression of the transcription factor Sp1 activates the OAS-RNAse L-RIG-I pathway.
Authors: Dupuis-Maurin V, Brinza L, Baguet J, Plantamura E, Schicklin S, Chambion S, Macari C, Tomkowiak M, Deniaud E, Leverrier Y, Marvel J, Michallet M
PLoS ONE, 2015-03-04;10(3):e0118551.
Species: Mouse
Sample Types: Cell Culture Supernates
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The role of platelet factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury.
Authors: Lapchak PH, Ioannou A, Rani P
PLoS ONE, 2012-07-06;7(7):e39934.
Species: Mouse
Sample Types: Plasma
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Pleiotropic platelet defects in mice with disrupted FOG1-NuRD interaction.
Authors: Wang Y, Meng R, Hayes V, Fuentes R, Yu X, Abrams CS, Heijnen HF, Blobel GA, Marks MS, Poncz M
Blood, 2011-10-11;118(23):6183-91.
Species: Mouse
Sample Types: Cell Culture Supernates
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Plasma proteome profiles associated with inflammation, angiogenesis, and cancer.
Authors: Kelly-Spratt KS, Pitteri SJ, Gurley KE, Liggitt D, Chin A, Kennedy J, Wong CH, Zhang Q, Buson TB, Wang H, Hanash SM, Kemp CJ
PLoS ONE, 2011-05-12;6(5):e19721.
Species: Mouse
Sample Types: Plasma
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In vivo platelet-endothelial cell interactions in response to major histocompatibility complex alloantibody.
Authors: Morrell CN, Murata K, Swaim AM, Mason E, Martin TV, Thompson LE, Ballard M, Fox-Talbot K, Wasowska B, Baldwin WM
Circ. Res., 2008-02-22;102(7):777-85.
Species: Mouse
Sample Types: Cell Culture Supernates
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Expression of CXCL4 in microglia in vitro and in vivo and its possible signaling through CXCR3.
Authors: de Jong EK, de Haas AH, Brouwer N, van Weering HR, Hensens M, Bechmann I, Pratley P, Wesseling E, Boddeke HW, Biber K
J. Neurochem., 2008-02-01;105(5):1726-36.
Species: Mouse
Sample Types: Cell Culture Supernates
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Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice.
Authors: Woulfe DS, Lilliendahl JK, August S, Rauova L, Kowalska MA, Abrink M, Pejler G, White JG, Schick BP
Blood, 2007-12-19;111(7):3458-67.
Species: Mouse
Sample Types: Serum
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Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release.
Authors: Schraw TD, Rutledge TW, Crawford GL, Bernstein AM, Kalen AL, Pessin JE, Whiteheart SW
Blood, 2003-05-08;102(5):1716-22.
Species: Mouse
Sample Types: Cell Culture Supernates
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Mouse blood was spun at 1000 RPM for 5:00 minutes, platelet rich plasma was removed, blood was spun at 3000 RPM for 5:00 minutes and plasma was removed from top for ELISA samples. Plasma was diluted 1:1000 into reagent diluent.
