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PE-Cy™7 Rat Anti-Mouse CD4
PE-Cy™7 Rat Anti-Mouse CD4

Multicolor flow cytometric analysis of CD4 expression on mouse splenocytes. Mouse splenic leucocytes were stained with APC Hamster Anti-Mouse CD3e antibody (Cat. No. 553066/561826) and either PE-Cy7 Rat IgG2a, κ Isotype Control (Cat. No. 552784; Left Plot) or PE-Cy7 Rat Anti-Mouse CD4 (Cat. No. 552775/561099; Right Plot) at 0.06 µg/test. The bivariate pseudocolor density plot showing the correlated expression of CD4 (or Ig Isotype control staining) versus CD3ε was derived from gated events with the forward and side light-scatter characteristics of intact leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Multicolor flow cytometric analysis of CD4 expression on mouse splenocytes. Mouse splenic leucocytes were stained with APC Hamster Anti-Mouse CD3e antibody (Cat. No. 553066/561826) and either PE-Cy7 Rat IgG2a, κ Isotype Control (Cat. No. 552784; Left Plot) or PE-Cy7 Rat Anti-Mouse CD4 (Cat. No. 552775/561099; Right Plot) at 0.06 µg/test. The bivariate pseudocolor density plot showing the correlated expression of CD4 (or Ig Isotype control staining) versus CD3ε was derived from gated events with the forward and side light-scatter characteristics of intact leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Product Details
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BD Pharmingen™
Cd4; CD4 antigen; L3T4; Ly-4; T-cell surface antigen T4/Leu-3
Mouse (QC Testing)
Rat DA, also known as DA/HA IgG2a, κ
Mouse Thymocytes (BALB/c)
Flow cytometry (Routinely Tested)
0.2 mg/ml
12504
AB_394461
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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The antibody was conjugated to the dye under optimum conditions and unconjugated antibody and free dye were removed.

Recommended Assay Procedures

BD® CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. 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.
  5. 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.
  6. 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.
  7. 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).
  8. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  9. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
552775 Rev. 7
Antibody Details
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RM4-5

The RM4-5 monoclonal antibody specifically binds to the CD4 (L3T4) differentiation antigen expressed on most thymocytes, subpopulations of mature T lymphocytes (i.e., MHC class II-restricted T cells, including most T helper cells and immunosuppressive regulatory T cells), and a subset of NK-T cells. CD4 has also been reported to be detected on pluripotent hematopoietic stem cells, bone marrow myeloid and B-lymphocyte precursors, intrathymic lymphoid precursors, and a subset of splenic dendritic cells. CD4 has been reported to be expressed on the plasma membrane of mouse egg cells and is involved in adhesion of the egg to MHC class II-bearing sperm. CD4 is an antigen coreceptor on the T-cell surface which interacts with MHC class II molecules on antigen-presenting cells. It participates in T-cell activation through its association with the T-cell receptor complex and protein tyrosine kinase lck. Purified RM4-5 mAb has been reported to block the binding of FITC-conjugated anti-mouse CD4 clones GK1.5 and H129.19, but not the RM4-4 clone.

552775 Rev. 7
Format Details
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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.
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PE-Cy7
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
781 nm
552775 Rev.7
Citations & References
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Development References (16)

  1. Allman D, Li J, Hardy RR. Commitment to the B lymphoid lineage occurs before DH-JH recombination. J Exp Med. 1999; 189(4):735-740. (Biology). View Reference
  2. Bendelac A. Mouse NK1+ T cells. Curr Opin Immunol. 1995; 7(3):367-374. (Biology). View Reference
  3. Bierer BE, Sleckman BP, Ratnofsky SE, Burakoff SJ. The biologic roles of CD2, CD4, and CD8 in T-cell activation. Annu Rev Immunol. 1989; 7:579-599. (Biology). View Reference
  4. Bosselut R, Zhang W, Ashe JM, Kopacz JL, Samelson LE, Singer A. Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction. J Exp Med. 1999; 190(10):1517-1526. (Biology: Immunoprecipitation). View Reference
  5. Brady BL, Rupp LJ, Bassing CH. Requirement for dicer in survival of proliferating thymocytes experiencing DNA double-strand breaks.. J Immunol. 2013; 190(7):3256-66. (Clone-specific: Flow cytometry). View Reference
  6. Frederickson GG, Basch RS. L3T4 antigen expression by hemopoietic precursor cells. J Exp Med. 1989; 169(4):1473-1478. (Biology). View Reference
  7. Godfrey DI, Kennedy J, Mombaerts P, Tonegawa S, Zlotnik A. Onset of TCR-β gene rearrangement and role of TCR-β expression during CD3-CD4-CD8- thymocyte differentiation. J Immunol. 1994; 152(10):4783-4792. (Biology). View Reference
  8. Guo MW, Watanabe T, Mori E, Mori T. Molecular structure and function of CD4 on murine egg plasma membrane. Zygote. 1995; 3(1):65-73. (Biology). View Reference
  9. Janeway CA Jr. The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu Rev Immunol. 1992; 10:645-674. (Biology). View Reference
  10. Martin P, del Hoyo GM, Anjuere F, et al. Concept of lymphoid versus myeloid dendritic cell lineages revisited: both CD8alpha(-) and CD8alpha(+) dendritic cells are generated from CD4(low) lymphoid-committed precursors. Blood. 2000; 96(7):2511-2519. (Biology). View Reference
  11. Nakamura T. Personal Communication. .
  12. Roederer M, Kantor AB, Parks DR, Herzenberg LA. Cy7PE and Cy7APC: bright new probes for immunofluorescence. Cytometry. 1996; 24(3):191-197. (Methodology: Flow cytometry). View Reference
  13. Shevach EM. Regulatory T cells in autoimmmunity. Annu Rev Immunol. 2000; 18:423-449. (Biology). View Reference
  14. Wineman JP, Gilmore GL, Gritzmacher C, Torbett BE, Muller-Sieburg CE. CD4 is expressed on murine pluripotent hematopoietic stem cells. Blood. 1992; 180(7):1717-1724. (Biology). View Reference
  15. Wu L, Antica M, Johnson GR, Scollay R, Shortman K. Developmental potential of the earliest precursor cells from the adult mouse thymus. J Exp Med. 1991; 174(6):1617-1627. (Biology). View Reference
  16. Wu L, Scollay R, Egerton M, Pearse M, Spangrude GJ, Shortman K. CD4 expressed on earliest T-lineage precursor cells in the adult murine thymus. Nature. 1991; 349(6304):71-74. (Biology). View Reference
View All (16) View Less
552775 Rev. 7

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