Mouse Flt-3 Ligand/FLT3L 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 Flt-3 Ligand. 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: Flt-3 Ligand/FLT3L
Flt-3 Ligand, also known as FL, is an alpha-helical cytokine that promotes the differentiation of multiple hematopoietic cell lineages. Mature human Flt-3 Ligand consists of a 158 amino acid (aa) extracellular domain (ECD) with a cytokine-like domain and a juxtamembrane tether region, a 21 aa transmembrane segment, and a 30 aa cytoplasmic tail. Within the ECD, human Flt-3 Ligand shares 71% and 65% aa sequence identity with mouse and rat Flt-3 Ligand, respectively. Human and mouse Flt-3 Ligand show cross-species activity. Flt-3 Ligand is expressed as a noncovalently-linked dimer by T cells and bone marrow and thymic fibroblasts. Each 36 kDa chain carries approximately 12 kDa of N- and O-linked carbohydrates. Alternate splicing and proteolytic cleavage of the transmembrane form can generate a soluble 30 kDa fragment that includes the cytokine domain. Alternate splicing of human Flt-3 Ligand also generates membrane-associated isoforms that contain either a truncated cytoplasmic tail or an 85 aa substitution following the cytokine domain. Both transmembrane and soluble Flt-3 Ligand signal through the tyrosine kinase receptor Flt-3/Flk-2. Flt-3 Ligand induces the expansion of monocytes and immature dendritic cells as well as early B cell lineage differentiation. It synergizes with IL-3, GM-CSF, and SCF to promote the mobilization and myeloid differentiation of hematopoietic stem cells. It cooperates with IL-2, -6, -7, and -15 to induce NK cell development and with IL-3, -7, and -11 to induce terminal B cell maturation. Animal studies also show Flt-3 Ligand to reduce the severity of experimentally induced allergic inflammation.
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 Flt-3 Ligand/FLT3L 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.
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Citations: Showing 1 - 5
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Long-term engraftment of adult hematopoietic progenitors in a novel model of humanized mice
Authors: Yu, CI;Maser, R;Marches, F;Banchereau, J;Palucka, K;
bioRxiv : the preprint server for biology
Species: Mouse
Sample Types: Plasma
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Guidelines for mouse and human DC generation
Authors: MB Lutz, S Ali, C Audiger, SE Autenrieth, L Berod, V Bigley, L Cyran, M Dalod, J Dörrie, D Dudziak, G Flórez-Gra, L Giusiano, GJ Godoy, M Heuer, AB Krug, CHK Lehmann, CT Mayer, SH Naik, S Scheu, G Schreibelt, E Segura, K Seré, T Sparwasser, J Tel, H Xu, M Zenke
European Journal of Immunology, 2022-10-27;0(0):.
Species: Mouse
Sample Types: Cell Culture Supernates
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Breast and pancreatic cancer interrupt IRF8-dependent dendritic cell development to overcome immune surveillance
Authors: MA Meyer, JM Baer, BL Knolhoff, TM Nywening, RZ Panni, X Su, KN Weilbaeche, WG Hawkins, C Ma, RC Fields, DC Linehan, GA Challen, R Faccio, RL Aft, DG DeNardo
Nat Commun, 2018-03-28;9(1):1250.
Species: Mouse
Sample Types: Serum
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Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand.
Authors: Chen Y, Chang S, Chen T, Lee C
PLoS ONE, 2015-08-11;10(8):e0135217.
Species: Mouse
Sample Types: Serum
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Activation of Fms-like tyrosine kinase 3 signaling enhances survivin expression in a mouse model of rheumatoid arthritis.
Authors: Andersson S, Svensson M, Erlandsson M, Dehlin M, Andersson K, Bokarewa M
PLoS ONE, 2012-10-17;7(10):e47668.
Species: Mouse
Sample Types: Cell Culture Supernates
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