Description
The 2D7/CCR5 monoclonal antibody specifically binds to the human chemokine receptor CCR5, also known as CD195. CCR5 is a seven transmembrane-spanning G protein-associated molecule that belongs to the beta chemokine receptor family and expressed on a subset of T lymphocytes (CD3+CD45RO+CD95+). CCR5 regulates lymphocyte chemotaxis activation and transendothelial migration during inflammation by signaling a response to at least three chemokines: Regulated upon Activation Normal T-cell Expressed and Secreted (RANTES), Macrophage Inflammatory Protein-1 (MIP-1), and Monocyte Chemoattractant Protein 2 (MCP-2). Additionally, CCR5 has been found to be a coreceptor for macrophage-tropic HIV-1 on CD4+ cells, a characteristic that is important in viral transmission. Reports indicate that individuals who have partial (heterozygous) or complete (homozygous) deletion of the CCR5 allele demonstrate resistance to HIV infection. This antibody has been shown to block ligand and gp120 binding. It is also able to neutralize HIV infection.
Immunophenotyping studies of chemokine receptors need to be performed on freshly collected whole blood (<24 Hrs). Incubation with the antibody should be done at room temperature in the dark. Cellular manipulation, such as Ficoll-Paque™ separation, freezing, or exposure to cold temperatures prior to staining have been shown to cause a decrease in staining intensity and inconsistent results.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with PE-Cy5 (formerly known as BD Cy-Chrome™) under optimum conditions, and unconjugated antibody and free PE-Cy5 were removed.
1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
2. PE-Cy5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the PE-Cy5 tandem fluorochrome, extra care must be taken when using dual-laser cytometers which may directly excite both PE and Cy5™.
3. Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
4. PE-Cy5 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by the 488 nm light of an Argon ion laser and serves as an energy donor, coupled to the cyanine dye Cy5, which acts as an energy acceptor and fluoresces at 670 nm. BD Biosciences Pharmingen has maximized the fluorochrome energy transfer in PE-Cy5, thus maximizing its fluorescence emission intensity, minimizing residual emission from PE, and minimizing lot-to-lot variation.
5. This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
7. 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.
8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
9. An isotype control should be used at the same concentration as the antibody of interest.
10. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
11. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
12. PE-Cy5 tandem fluorochromes have been reported to bind some classes of human macrophages and granulocytes via Fc receptors, and PE has been reported to bind to mouse B lymphocytes via Fc receptors. Preincubation of mouse leukocytes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 can reduce the non-specific binding of PE-Cy5-conjugated reagents to mouse B cells. However, PE-Cy5 conjugated reagents should not be used to stain splenocytes of SJL, NOD, and MRL mice as B lymphocytes and/or other leukocytes have been reported to non-specifically stain regardless of the use of Mouse BD Fc Block™ (the CD72c complex has been implicated for PE-Cy5 binding in these strains). Reagents conjugated to PE, PerCP, PerCP-Cy5.5, APC, and APC-Cy7 tandem fluorochrome can be used on leukocytes from these mouse strains.
13. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
