Staining of 3-day Anti-Mouse CD3 (cat. 16-0031) and Anti-Mouse CD28 (cat. 16-0281)-stimulated C57BL/6 splenocytes with 0.25 µg of Rat IgG1 κ Isotype Control Purified (cat. 14-4301) (open histogram) or 0.25 µg of Anti-Mouse CD223 (Lag-3) Purified (filled histogram) followed by Anti-Rat IgG Biotin (cat. 13-4813) and Streptavidin PE (cat. 12-4317). Total viable cells were used for analysis.
Contents: Anti-Mouse CD223 (Lag-3) Purified Catalog Number: 14-2231 Concentration: 0.5 mg/ml Formulation: Phosphate buffer pH 7.2, 150 mM NaCl, 0.09% NaN3 Storage Conditions: Store at 2-8°C. Clone: eBioC9B7W (C9B7W) Host/Isotype: Rat IgG1, κ
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Cat. No.
Size
Price
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14-2231-81
50 ug
14-2231-82
100 ug
14-2231-85
500 ug
Note: Several countries will continue to be supplied via distributors. Country specific prices may apply.
*Functional Grade™ (FG™) Purified: Azide-free, sterile-filtered, and endotoxin < 0.001 ng/µg (unless otherwise noted). *Functional Grade™ (FG™) Biotin: Azide-free, sterile-filtered, and endotoxin < 0.05 ng/µg. Purified: Contains azide, not sterile-filtered, and not endotoxin tested.
Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Alexa Fluor® 647 conjugated products with eFluor® 660 conjugated products.
Description
The eBioC9B7W monoclonal antibody recognizes mouse CD223 (LAG-3, LAG3) protein expressed by activated αβ-TCR bearing T cells, a subset of γδ-TCR bearing T cells and a subset of NK cells. CD223 is a 70 kDa type I transmembrane protein with a structure that is similar to CD4. However, a soluble form of human CD223 has been detected by ELISA in human serum, and data suggest that mouse CD223 is proteolytically cleaved in the D4 domain. This results in a 54 kDa fragment containing all the extracellular domains, and a 16 kDa fragment containing the intracellular and transmembrane domains. The 54 kDa can remain membrane-associated or be released as soluble CD223.
CD223 binds to MHC class II with higher affinity than CD4, and it is thought that this interaction is involved in the negative regulation of T-cell activation and homeostatic proliferation. Furthermore, CD223 is expressed by CD4+CD25+ regulatory T cells, and it has been suggested that CD223 may be involved in their regulatory function.
Applications Reported
For research use only, not for diagnostic or therapeutic use. This eBioC9B7W (C9B7W) antibody has been reported for use in flow cytometric analysis, immunoprecipitation, and ELISA. (Please use Functional Grade purified eBioC9B7W (C9B7W), cat. 16-2231, in functional assays.)
Applications Tested
This eBioC9B7W (C9B7W) antibody has been tested by flow cytometric analysis of anti-CD3 and anti-CD28-activated mouse splenocytes. This can be used at less than or equal to 0.5 μg per test. A test is defined as the amount (μg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 105 to 108 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
This eBioC9B7W (C9B7W) antibody has also been tested by ELISA analysis of supernatant produced from mouse CD223-transfected 3A9 cells. This can be used in ELISA at 2 μg/ml for capture of the secreted form of CD223, together with the polyclonal anti-mouse CD223 (cat. 24-2232) as the detection antibody.
References
Baixeras E, Huard B, Miossec C, Jitsukawa S, Martin M, Hercend T, Auffray C, Triebel F, Piatier-Tonneau D.
Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.
J Exp Med. 1992 Aug 1;176(2):327-37.
Workman CJ, Rice DS, Dugger KJ, Kurschner C, Vignali DA.
Phenotypic analysis of the murine CD4-related glycoprotein, CD223 (LAG-3).
Eur J Immunol. 2002 Aug;32(8):2255-63. (C9B7W, FC, FA, PubMed)
Workman CJ, Vignali DA.
Negative regulation of T cell homeostasis by lymphocyte activation gene-3 (CD223).
J Immunol. 2005 Jan 15;174(2):688-95. (C9B7W, FA, PubMed)
Li N, Workman CJ, Martin SM, Vignali DA.
Biochemical analysis of the regulatory T cell protein lymphocyte activation gene-3 (LAG-3; CD223).
J Immunol. 2004 Dec 1;173(11):6806-12. (C9B7W, IP, PubMed)
Huang CT, Workman CJ, Flies D, Pan X, Marson AL, Zhou G, Hipkiss EL, Ravi S, Kowalski J, Levitsky HI, Powell JD, Pardoll DM, Drake CG, Vignali DA.
Role of LAG-3 in regulatory T cells.
Immunity. 2004 Oct;21(4):503-13. (C9B7W, FC, FA, PubMed)
