PE-Cy™5 Rat Anti-Mouse CD3 Molecular Complex
Clone 17A2 (RUO)
- Brand BD Pharmingen™
- Alternative Name CD3; CD3 epsilon; Cd3e; CD3ε; T3e
- Concentration 0.2 mg/ml
- Isotype Rat SD, also known as Sprague-Dawley (outbred) IgG2b, κ
- Reactivity Mouse (QC Testing)
Flow cytometry (Routinely Tested)
- Immunogen γδ TCR-positive T-T hybridoma D1
- Entrez Gene ID 12501
- Storage Buffer Aqueous buffered solution containing protein stabilizer and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The 17A2 monoclonal antibody specifically binds to the T-cell receptor-associated CD3 complex that is expressed on many thymocytes and mature T lymphocytes. Plate-bound 17A2 antibody has been reported to induce IL-2 production by cultured T cells in the absence of accessory cells. The binding of the 17A2 antibody to T cells can be blocked by the anti-CD3e mAb 145-2C11 (Cat. No. 557306/553058/550275). This suggests that the 17A2 antibody recognizes an epitope of the CD3 epsilon chain. In vivo treatment with 17A2 antibody has been reported to partially deplete T lymphocytes and temporarily down-modulates CD3 expression on T cells.
- Format PE-Cy™5
- Excitation Source Blue 488 nm,Green 532 nm,Yellow/Green 561 nm
- Excitation Max 496 nm
- Emission Max 667 nm
PE-Cy™5 is a tandem conjugate that combines phycoerythrin and a cyanine dye. Because of its broad absorption range and the fact that its emission spectra are equivalent to APC, PE-Cy5 is not recommended for simultaneous use with APC. The Cy5 molecule has been shown to exhibit nonspecific binding to Fc receptors, which is most apparent on monocyte populations. PE-Cy5 is not recommended for fluorescence microscopy because it is subject to photobleaching.
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Preparation and Storage
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.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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™.
- 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.
- Cy is a trademark of GE Healthcare.
- 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.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
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 (Cat. No. 553141/553142) can reduce the non-specific binding of PE-Cy5-conjugated reagents to mouse B cells.
Furthermore, we have observed a distinct interaction between PE-Cy5 tandem fluorochromes and the splenocytes of SJL, NOD, and MRL mouse: B lymphocytes and some other leukocyte subsets are brightly stained, and Mouse BD Fc Block™ has no significant effect. Therefore, PE-Cy5-conjugated reagents should not be used to stain leukocytes of SJL, NOD, or MRL mice. Reagents conjugated to PE, PerCP, PerCP-Cy5.5, Allophycocyanin (APC), and APC-Cy7 tandem fluorochrome can be used on leukocytes from these mouse strains.