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BUV661 Rat Anti-Mouse CD4
BUV661 Rat Anti-Mouse CD4

Two color flow cytometric analysis of CD4 expression on mouse splenocytes. Mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/32 antibody (Mouse Fc Block™) (Cat. No. 553141/553142). The cells were then stained with FITC Hamster Anti-Mouse CD3e antibody (Cat. No. 553061/553062/561827) and either BD Horizon™ BUV661 Rat IgG2b, κ Isotype Control (Cat. No. 612978; Left Plot) or BD Horizon™ BUV661 Rat Anti-Mouse CD4 antibody (Cat. No. 612974; Right Plot) at 0.5 µg/test. The two-color pseudocolor density plot showing the correlated expression of CD4 (or Ig Isotype Control staining) versus CD3e was derived from gated events with the forward and side light-scatter characteristic of viable splenic leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Two color flow cytometric analysis of CD4 expression on mouse splenocytes. Mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/32 antibody (Mouse Fc Block™) (Cat. No. 553141/553142). The cells were then stained with FITC Hamster Anti-Mouse CD3e antibody (Cat. No. 553061/553062/561827) and either BD Horizon™ BUV661 Rat IgG2b, κ Isotype Control (Cat. No. 612978; Left Plot) or BD Horizon™ BUV661 Rat Anti-Mouse CD4 antibody (Cat. No. 612974; Right Plot) at 0.5 µg/test. The two-color pseudocolor density plot showing the correlated expression of CD4 (or Ig Isotype Control staining) versus CD3e was derived from gated events with the forward and side light-scatter characteristic of viable splenic leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Product Details
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BD Horizon™
Cd4; CD4 antigen; L3T4; Ly-4; T-cell surface antigen T4/Leu-3
Mouse (QC Testing)
Rat LEW, also known as Lewis IgG2b, κ
Mouse CTL clone V4
Flow cytometry (Routinely Tested)
0.2 mg/ml
12504
AB_2870246
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


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 BD Horizon BUV661 under optimum conditions, and unconjugated antibody and free BD Horizon BUV661 were removed.

Recommended Assay Procedures

     BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation).  When fluorochrome conjugated antibodies are bound to 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 CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.

     For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment.  Fluorescent dye interactions may cause staining artifacts which may affect data interpretation.  The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions.  More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).

     For optimal results, it is recommended to perform 2 washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescence staining, prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. BD Horizon Brilliant Ultraviolet 661 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,575,303; 8,354,239.
  6. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
  7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
612974 Rev. 1
Antibody Details
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GK1.5

The GK1.5 monoclonal antibody specifically binds to the mouse CD4 (L3T4) differentiation antigen. CD4 is expressed on most thymocytes, a subpopulation of mature T lymphocytes (i.e., MHC class II-restricted T cells, including most T helper cells), and a subset of NK-T cells. In addition, CD4 has also been reported to be detectable on pluripotent hematopoietic stem cells, bone marrow myeloid and B-lymphocyte precursors, intrathymic lymphoid precursors, and a subset of splenic dendritic cells. CD4 has also 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. The GK1.5 antibody reportedly blocks binding of the RM4-5 and H129.19, but not RM4-4 mouse CD4-specific antibodies.

The antibody was conjugated to BD Horizon BUV661 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome of BD Horizon BUV395 with an Ex Max of 348-nm and an acceptor dye with an Em Max at 661-nm. BD Horizon Brilliant BUV661 can be excited by the ultraviolet laser (355 nm) and detected with a 670/25 filter and a 630 nm LP.  Due to cross laser excitation of this dye, there may be significant spillover into channels detecting APC-like emissions (eg, 670/25-nm filter).

612974 Rev. 1
Format Details
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BUV661
The BD Horizon Brilliant™ Ultraviolet 661 (BUV661) Dye is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This tandem fluorochrome is comprised of a BUV395 donor with an excitation maximum (Ex Max) of 350-nm and an acceptor dye with an emission maximum (Em Max) at 660-nm. BUV661, driven by BD innovation, is designed to be excited by the ultraviolet laser (355-nm) and detected using an optical filter centered near 660-nm (e.g., 670/25 bandpass filter). The acceptor dye can be excited by the Red (628–640-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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BUV661
Ultraviolet 355 nm
350 nm
660 nm
612974 Rev.1
Citations & References
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Development References (10)

  1. 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. (Clone-specific: (Co)-stimulation, Immunoprecipitation). View Reference
  2. Dialynas DP, Quan ZS, Wall KA, et al. Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule. J Immunol. 1983; 131(5):2445-2451. (Immunogen: Blocking, Depletion, Flow cytometry). View Reference
  3. Dialynas DP, Wilde DB, Marrack P, et al. Characterization of the murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK1.5: expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHC antigen-reactivity. Immunol Rev. 1983; 74:29-56. (Clone-specific: Blocking, Depletion, Flow cytometry). View Reference
  4. Frederickson GG, Basch RS. L3T4 antigen expression by hemopoietic precursor cells. J Exp Med. 1989; 169(4):1473-1478. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
  5. Ghobrial RR, Boublik M, Winn HJ, Auchincloss H Jr. In vivo use of monoclonal antibodies against murine T cell antigens. Clin Immunol Immunopathol. 1989; 52(3):486-506. (Biology). View Reference
  6. 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
  7. Kruisbeek AM. In vivo depletion of CD4- and CD8-specific T cells. Curr Protoc Immunol. 1991; 4.1.1-4.1.5. (Clone-specific: Cytotoxicity, Depletion). View Reference
  8. 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. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting, Immunofluorescence, Immunoprecipitation). View Reference
  9. 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
  10. Zheng B, Han S, Kelsoe G. T helper cells in murine germinal centers are antigen-specific emigrants that downregulate Thy-1. J Exp Med. 1996; 184(3):1083-1091. (Clone-specific: Immunohistochemistry). View Reference
View All (10) View Less
612974 Rev. 1

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.