BD Pharmingen™

Technical Data Sheet Purified Rat Anti-Mouse CD200

Product Information Material Number: 552512 Alternate Name: OX-2 Antigen Size: 0.5 mg Concentration: 0.5 mg/ml Clone: OX-90 Immunogen: Soluble fusion protein of the extracellular region of mouse OX-2 antigen w/domains 3 & 4 of rat CD4 Isotype: Rat IgG2a, κ Reactivity: QC Testing: Mouse Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide. Description The OX-90 antibody reacts with CD200 (OX-2 antigen), a type-1 membrane glycoprotein containing two extracellular Ig-like domains. CD200 is expressed on thymocytes, neurons, B lymphocytes, splenic follicular dendritic cells and endothelium, and subsets of T lymphocytes and dendritic cells; but not on NK cells, granulocytes, monocytes, or macrophages. The CD200 Receptor is restricted to myeloid cells, and it is believed that its engagement with CD200 results in inhibition and/or down-regulation of myeloid cell activity.

Multi-color analysis of the expression of CD200 on thymocytes. C57BL/6 thymocytes were stained with either purified mAb OX-90 (open histograms) or purified rat IgG2a, κ isotype control mAb R35-95 (Cat. No. 553927, shaded histograms), followed by biotinylated anti-rat IgG2a mAb RG7/1.30 (Cat. No. 553894) then Streptavidin-PE (Cat. No. 554061). Then the cells were stained with FITC-conjugated anti-mouse CD4 mAb and APC-conjugated anti-mouse CD8a mAb 53-6.7 (Cat. No. 553035). Electronic gates were set to include the CD4-CD8a-, CD4+CD8a+, CD4+CD8a-, and CD4-CD8a+ populations, and their expression of CD200 is displayed in the corresponding panels. Flow cytometry was performed on a BD FACSCalibur™ Flow Cytometry System.

Preparation and Storage The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C.

552512 Rev. 1 Page 1 of 2 Application Notes Application Flow cytometry Routinely Tested ELISA Reported Immunohistochemistry Reported

Recommended Assay Procedure: Since this antigen is expressed at low density on resting peripheral leukocytes, it may be desirable to amplify staining by using a biotinylated second-step antibody followed by a "bright" third-step reagent, such as Streptavidin-PE (Cat. No. 554061). We also recommend the use of Mouse BD Fc Block™ (anti-mouse CD16/CD32, mAb 2.4G2, Cat. No. 553141/553142) when Fc Receptor-bearing cells are present. For staining with purified antibody in the presence of Mouse BD Fc Block™, a second-step antibody which does not react with the 2.4G2 antibody (Rat IgG2b, κ) must be used. We have found that biotinylated anti-rat IgG2a mAb RG7/1.30 (Cat. No. 553894) is useful.

Suggested Companion Products

Catalog Number Name Size Clone 553927 Purified Rat IgG2a, κ Isotype Control 0.5 mg R35-95 553894 Biotin Mouse Anti-Rat IgG2a 0.5 mg RG7/1.30 554061 PE Streptavidin 0.5 mg (none) 553035 APC Rat Anti-Mouse CD8a 0.1 mg 53-6.7 553141 Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) 0.1 mg 2.4G2

Product Notices 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results. 2. Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols. 3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing. 4. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.

References Barclay NA, Brown MH, Birkeland ML, et al, ed. The Leukocyte Antigen FactsBook. San Diego, CA: Academic Press; 1997.(Biology) Gorczynski L, Chen Z, Hu J, et al. Evidence that an OX-2-positive cell can inhibit the stimulation of type 1 cytokine production by bone marrow-derived B7-1 (and B7-2)-positive dendritic cells. J Immunol. 1999; 162(2):774-781.(Biology) Hoek RM, Ruuls SR, Murphy CA, et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science. 2000; 290(5497):1768-1771. (Immunogen: ELISA, Immunohistochemistry) Preston S, Wright GJ, Starr K, Barclay AN, Brown MH. The leukocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages. Eur J Immunol. 1997; 27(8):1911-1918.(Immunogen)

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