Multicolor flow cytometric analysis of CD4 expression on pig peripheral blood lymphocytes. Pig whole blood was stained simultaneously with PE-Cy™7 Mouse Anti-Pig CD4a antibody (Cat. No. 561473) and FITC Mouse Anti-Pig CD3ε antibody (Cat. No. 559582). The erythrocytes were lysed with BD PharmLyse™ Lysing Buffer (Cat. No. 555899). Two-color flow cytometric dot plots showing the correlated expression of CD4a versus CD3 were derived from gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
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Multicolor flow cytometric analysis of CD4 expression on pig peripheral blood lymphocytes. Pig whole blood was stained simultaneously with PE-Cy™7 Mouse Anti-Pig CD4a antibody (Cat. No. 561473) and FITC Mouse Anti-Pig CD3ε antibody (Cat. No. 559582). The erythrocytes were lysed with BD PharmLyse™ Lysing Buffer (Cat. No. 555899). Two-color flow cytometric dot plots showing the correlated expression of CD4a versus CD3 were derived from gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Pharmingen™
CD4; CD4 molecule; Lymphocyte antigen CD4
Pig (QC Testing)
Mouse BALB/c IgG2b, κ
dd miniature swine thymocytes
Flow cytometry (Routinely Tested)
0.2 mg/ml
AB_10683164
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Recommended Assay Procedures
PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. PE-Cy7-labeled antibodies can be used with FITC- and R-PE-labeled reagents in single-laser flow cytometers with no significant spectral overlap between PE-Cy7 and FITC.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- 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.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
- 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.
- PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Lysing Buffer RUO
Size
100 mL
Cat No.
555899
FITC Mouse Anti-Pig CD3ε RUO
Size
0.1 mg
Cat No.
559582
561473 Rev. 1
Antibody Details
74-12-4
The 74-12-4 (also known as clone PT4) monoclonal antibody specifically binds to CD4, a 55-kDa antigen expressed on T lymphocytes. This antibody does not react with CTL effectors, CTL precursors, or NK cells (ie, CD8[bright] cells) and it does not cross-react with human or bovine cells. Two peripheral T-helper lymphocyte phenotypes can be distinguished in the pig: CD4+CD8- and CD4+CD8[dull]. mAb 74-12-4 has been reported to inhibit proliferative responses of peripheral blood lymphocytes to mitogen, soluble antigen, and alloantigen. It is only marginally effective for in vivo depletion of peripheral CD4+ T cells. Two alloantigenic forms of CD4 have been recognized in miniature swine based upon their recognition (CD4.1) or lack of recognition (CD4.2) by mAb 74-12-4; the CD4.2 phenotype displays an autosomal recessive, non-MHC-linked, pattern of inheritance. The molecular basis for the polymorphism is a cluster of nucleotide differences leading to multiple amino-acid substitutions in the Ig CDR2-like loop structure. This mAb was clustered as anti-CD4a at the First International Swine CD Workshop. It has been reported to crossreact with chicken leukocytes.
561473 Rev. 1
Format Details
PE-Cy7
PE-Cy7 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 781-nm. PE can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 781 nm (e.g., a 760/60-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the Red (627–640-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PE-Cy7
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
781 nm
561473 Rev.1
Citations & References
Development References (10)
-
Brodersen R, Bijlsma F, Gori K. Analysis of the immunological cross reactivities of 213 well characterized monoclonal antibodies with specificities against various leucocyte surface antigens of human and 11 animal species. Vet Immunol Immunopathol. 1998; 64(1):1-13. (Biology). View Reference
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Dato ME, Kim YB. Characterization and utilization of a monoclonal antibody inhibiting porcine natural killer cell activity for isolation of natural killer and killer cells. J Immunol. 1990; 144(11):4452-4462. (Immunogen). View Reference
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Gustafsson K, Germana S, Sundt TM, Sachs DH, LeGuern C. Extensive allelic polymorphism in the CDR2-like region of the miniature swine CD4 molecule. J Immunol. 1993; 151(3):1365-1370. (Biology). View Reference
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Pescovitz MD, Lunney JK, Sachs DH. Murine anti-swine T4 and T8 monoclonal antibodies: distribution and effects on proliferative and cytotoxic T cells. J Immunol. 1985; 134(1):37-44. (Biology). View Reference
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Pescovitz MD, Lunney JK, Sachs DH. Preparation and characterization of monoclonal antibodies reactive with porcine PBL. J Immunol. 1984; 133(1):368-375. (Biology). View Reference
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Saalmuller A, Aasted B, Canals A, et al. Analyses of mAb reactive with porcine CD8. Vet Immunol Immunopathol. 1994; 43(1-3):249-254. (Biology). View Reference
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Saalmüller A, Aasted B, Canals A. Summary of workshop findings for porcine T-lymphocyte antigens. Vet Immunol Immunopathol. 1994; 43(1-3):219-228. (Biology). View Reference
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Smith CV, Sablinski T, Arn JS, et al. In vivo treatment with monoclonal antibodies directed against CD4 and CD8 antigens in miniature swine. J Immunother Emphasis Tumor Immunol. 1994; 16(2):105-114. (Biology). View Reference
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Sundt TM, LeGuern C, Germana S. Characterization of a polymorphism of CD4 in miniature swine. J Immunol. 1992; 148(10):3195-3201. (Biology). View Reference
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Suzuki T, Sundt TM 3rd, Mixon A, Sachs DH. In vivo treatment with antiporcine T cell antibodies. Transplantation. 1990; 50(1):76-81. (Biology). View Reference
561473 Rev. 1
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
