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BB515 Rat Anti-Mouse I-A/I-E
BB515 Rat Anti-Mouse I-A/I-E

Two-color flow cytometric analysis of I-A/I-E MHC class II alloantigen expression on mouse splenocytes - Staining comparisons between BD Horizon™ BB515- and FITC-conjugated antibodies. C57BL/6 mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with APC Rat Anti-Mouse CD45R/B220 (Cat. No. 553092/561880) and APC Rat Anti-Mouse CD11b (Cat. No. 553312/561690) antibodies, and either BB515 Rat IgG2a, κ Isotype Control (Cat. No. 564418; Left Panel), BD Horizon BB515 Rat Anti-Mouse I-A/I-E (Cat. No. 565254; Middle Panel), or FITC Rat Anti-Mouse I-A/I-E (Cat. No. 553623/562009; Right Panel). Two-color flow cytometric contour plots showing the expression of I-A/I-E MHC class II alloantigens versus CD45R/B220 and CD11b were derived from gated events with the forward and side light-scatter characteristics of viable leucocytes. Mouse splenic leucocytes from SJL mice (H-2s haplotype) did not stain positively with the 2G9 antibody (data not shown). Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

Two-color flow cytometric analysis of I-A/I-E MHC class II alloantigen expression on mouse splenocytes - Staining comparisons between BD Horizon™ BB515- and FITC-conjugated antibodies. C57BL/6 mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with APC Rat Anti-Mouse CD45R/B220 (Cat. No. 553092/561880) and APC Rat Anti-Mouse CD11b (Cat. No. 553312/561690) antibodies, and either BB515 Rat IgG2a, κ Isotype Control (Cat. No. 564418; Left Panel), BD Horizon BB515 Rat Anti-Mouse I-A/I-E (Cat. No. 565254; Middle Panel), or FITC Rat Anti-Mouse I-A/I-E (Cat. No. 553623/562009; Right Panel). Two-color flow cytometric contour plots showing the expression of I-A/I-E MHC class II alloantigens versus CD45R/B220 and CD11b were derived from gated events with the forward and side light-scatter characteristics of viable leucocytes. Mouse splenic leucocytes from SJL mice (H-2s haplotype) did not stain positively with the 2G9 antibody (data not shown). Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

Product Details
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BD Horizon™
H-2I; Ia Ag; I-Ad, I-Ed, I-Eb, and I-Ek MHC class II alloantigens
Mouse (QC Testing)
Rat DA, also known as DA/HA IgG2a, κ
BALB/c mouse epidermal Langerhans cells
Flow cytometry (Routinely Tested)
0.2 mg/ml
AB_2739134
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™ BB515 under optimum conditions and unconjugated antibody was 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. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. Diagnostic uses require a separate license.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  7. 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.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
565254 Rev. 2
Antibody Details
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2G9

The 2G9 monoclonal antibody reacts with the I-Ad and I-Ed MHC class II alloantigens.  It also reacts with transfectants expressing I-Eb and I-Ek and with cells from mice of the H-2p and H-2q haplotypes.  This antibody is non-reactive with cells from SJL (H-2s) and NOD (H-2g7) mice.  Flow cytometric analysis indicates that the 2G9 and M5/114.15.2 monoclonal antibodies have comparable reactivity on cells from mice with I-Ab, I-Ad, I-Aq, I-Ed, and I-Ek alloantigens.

The antibody was conjugated to BD Horizon BB515 which is part of the BD Horizon Brilliant™ Blue family of dyes. With an Ex Max near 490 nm and an Em Max near 515 nm, BD Horizon BB515 can be excited by the blue laser (488 nm) laser and detected with a 530/30 nm filter. This dye has been exclusively developed by BD Biosciences and is up to seven times brighter than FITC with less spillover into the PE channel. Due to similar excitation and emission properties, BB515, FITC, and Alexa Fluor® 488 cannot be used simultaneously. It is not recommended to use BB515 in cocktails that include Streptavidin conjugates as it may cause high background.

565254 Rev. 2
Format Details
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BB515
The BD Horizon Brilliant™ Blue 515 (BB515) dye is part of the BD Horizon Brilliant™ Blue family of dyes. This dye is a polymer fluorochrome with an excitation maximum (Ex Max) at 490-nm and an emission maximum (Em Max) of 515-nm. Driven by BD innovation, BB515 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 520-nm (e.g., 530/30-nm). BB515 reagents are significantly brighter than equivalent FITC or Alexa Fluor™ 488 reagents with less spillover into the PE detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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BB515
Blue 488 nm
490 nm
515 nm
565254 Rev.2
Citations & References
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Development References (2)

  1. Becker D, Mohamadzadeh M, Reske K, Knop J. Increased level of intracellular MHC class II molecules in murine Langerhans cells following in vivo and in vitro administration of contact allergens. J Invest Dermatol. 1992; 99(5):545-549. (Immunogen: Immunofluorescence, Immunoprecipitation). View Reference
  2. Farquhar CA, Paterson AM, Cobbold SP, et al. Tolerogenicity is not an absolute property of a dendritic cell. Eur J Immunol. 2010; 40(6):1728-1737. (Clone-specific: Flow cytometry). View Reference
565254 Rev. 2

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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.