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RB744 Mouse Anti-Mouse H-2Kb/SIINFEKL

BD OptiBuild™ RB744 Mouse Anti-Mouse H-2Kb/SIINFEKL

Clone 25-D1.16.rMAb (also known as 25-D1.16)

Product Details
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BD OptiBuild™
Mouse (Tested in Development)
Mouse IgG1, κ
SIINFEKL-pulsed RMA-S cells
Flow cytometry (Qualified)
0.2 mg/ml
Aqueous buffered solution containing ≤0.09% sodium azide.

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 to the dye under optimum conditions that minimize unconjugated dye and antibody.

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. Please refer to for technical protocols.
  2. Please refer to to access safety data sheets (SDS).
  3. For U.S. patents that may apply, see
  4. 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.
  5. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  6. The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
  7. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit
  8. When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
  9. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at
  10. An isotype control should be used at the same concentration as the antibody of interest.
  11. Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
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Antibody Details
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25-D1.16.rMAb is a recombinant monoclonal antibody derived from 25-D1.16 hybridoma cells. The 25-D1.16.rMAb specifically recognizes the ovalbumin (OVA)-derived peptide SIINFEKL (amino acid residues 257-264 of OVA) bound to the MHC class l antigen, H-2Kb. It does not bind to either unbound H-2Kb or H-2Kb bound to an irrelevant peptide. The 25-D1.16 antibody has been found useful in a variety of in vitro and in vivo experimental model systems to study the nature of antigen-presenting cells that can present the SIINFEKL peptide in an MHC class I-restricted fashion to T cells. The 25-D1.16 monoclonal antibody can reportedly inhibit the T cell response to H-2Kb-SIINFEKL and be used for immunofluorescent or immunohistochemical staining of H-2Kb-SIINFEKL-positive cells.

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Format Details
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The BD Horizon RealBlue™ 744 (RB744) Dye is part of the BD® family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 746-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RB744 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), we recommend using an optical filter centered near 750-nm (e.g., a 750/60-nm bandpass filter).
Blue 488 nm
498 nm
746 nm
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Citations & References
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Development References (4)

  1. Dolan BP, Li L, Takeda K, Bennink JR, Yewdell JW. Defective ribosomal products are the major source of antigenic peptides endogenously generated from influenza A virus neuraminidase. J Immunol. 2010; 184(3):1419-1424. (Clone-specific: Flow cytometry, Fluorescence microscopy, Immunofluorescence). View Reference
  2. Hervé J, Dubreil L, Tardif V, et al. β2-Adrenoreceptor agonist inhibits antigen cross-presentation by dendritic cells.. J Immunol. 2013; 190(7):3163-71. (Clone-specific: Flow cytometry, Fluorescence microscopy, Immunofluorescence). View Reference
  3. Mareeva T, Wanjalla C, Schnell MJ, Sykulev Y. A novel composite immunotoxin that suppresses rabies virus production by the infected cells.. J Immunol Methods. 2010; 353(1-2):78-86. (Clone-specific: Cytotoxicity, Flow cytometry). View Reference
  4. Porgador A, Yewdell JW, Deng Y, Bennink JR, Germain RN. Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody.. Immunity. 1997; 6(6):715-26. (Immunogen: Flow cytometry, Fluorescence microscopy, Immunofluorescence, Immunohistochemistry). View Reference
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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.