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BV421 Rat Anti-Mouse I-A/I-E
BV421 Rat Anti-Mouse I-A/I-E

Multicolor flow cytometric analysis of I-A/I-E MHC class II alloantigen expression on splenocytes from positive and negative mouse strains (left and center panels). Mouse spleen cells from either M5/114-negative SJL (Left Panel) or M5/114-positive BALB/c (Center Panel) mice were stained with BD Horizon™ BV421 Rat Anti-Mouse I-A/I-E (Cat. No. 562564), APC Anti-Mouse CD45R/B220 (Cat. No. 553092/561880) and APC Anti-Mouse CD11b (Cat. No. 553312/561690) antibodies. Two-color flow cytometric dot 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 splenocytes. The M5/114 monoclonal antibody detects I-Ad and I-Ed MHC class II alloantigens that are expressed on both B cells and macrophages. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.

Immunohistofluorescent analysis of I-A/I-E expression by cells within C57BL/6 mouse spleen (right panel). A mouse spleen cryosection (5 µm) was fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), blocked with 5% goat serum and 1% BSA diluted in 1x PBS, and stained with BD Horizon™ BV421 Rat Anti-Mouse I-A/I-E antibody (Cat. No. 562564, pseudo-colored green) and Alexa Fluor® 647 Rat-anti-Mouse CD19 (Cat. No. 557684, pseudo-colored blue). Images were captured on a standard epifluorescence microscope. Original magnification, 20x.

Multicolor flow cytometric analysis of I-A/I-E MHC class II alloantigen expression on splenocytes from positive and negative mouse strains (left and center panels). Mouse spleen cells from either M5/114-negative SJL (Left Panel) or M5/114-positive BALB/c (Center Panel) mice were stained with BD Horizon™ BV421 Rat Anti-Mouse I-A/I-E (Cat. No. 562564), APC Anti-Mouse CD45R/B220 (Cat. No. 553092/561880) and APC Anti-Mouse CD11b (Cat. No. 553312/561690) antibodies. Two-color flow cytometric dot 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 splenocytes. The M5/114 monoclonal antibody detects I-Ad and I-Ed MHC class II alloantigens that are expressed on both B cells and macrophages. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.

Immunohistofluorescent analysis of I-A/I-E expression by cells within C57BL/6 mouse spleen (right panel). A mouse spleen cryosection (5 µm) was fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), blocked with 5% goat serum and 1% BSA diluted in 1x PBS, and stained with BD Horizon™ BV421 Rat Anti-Mouse I-A/I-E antibody (Cat. No. 562564, pseudo-colored green) and Alexa Fluor® 647 Rat-anti-Mouse CD19 (Cat. No. 557684, pseudo-colored blue). Images were captured on a standard epifluorescence microscope. Original magnification, 20x.

Product Details
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BD Horizon™
M5/114; H-2I; Ia Ag; I-Ab, I-Ad, I-Aq, I-Ed, and I-Ek MHC class II alloAgs
Mouse (QC Testing)
Rat BN x LEW IgG2b, κ
Activated C57BL/6 Mouse Spleen Cells
Flow cytometry (Routinely Tested), Immunofluorescence (Tested During Development)
0.2 mg/ml
AB_2716857
Aqueous buffered solution containing BSA and ≤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 BV421 under optimum conditions, and unconjugated antibody and free BD Horizon BV421 were removed.

Recommended Assay Procedures

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).

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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
  7. BD Horizon Brilliant Violet 421 is covered by one or more of the following US patents: 8,158,444; 8,362,193; 8,575,303; 8,354,239.
  8. 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.
  9. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
562564 Rev. 3
Antibody Details
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M5/114.15.2

The M5/114.15.2 monoclonal antibody recognizes a polymorphic determinant shared by the I-A[b], I-A[d], I-A[q], I-E[d], and I-E[k] (but not I-A[f], I-A[k], or I-A[s]) MHC class II alloantigens that can be expressed by B cells, dendritic cells, monocytes, macrophages and activated T cells. It also reacts with cells from mice of the H-2[p] and H-2[r] haplotypes, and it is non-reactive with cells from NOD (H-2[g7]) mice. Flow cytometric analysis indicates that the M5/114.15.2 and 2G9 monoclonal antibodies have comparable reactivity on cells from mice with I-A[b], I-A[d], I-A[g7], I-A[q], I-E[d], and I-E[k] alloantigens.

The antibody was conjugated to BD Horizon™ BV421 which is part of the BD Horizon Brilliant™ Violet family of dyes.  With an Ex Max of 407-nm and Em Max at 421-nm, BD Horizon™ BV421  can be excited by the violet laser and detected in the standard Pacific Blue™ filter set (eg, 450/50-nm filter). BD Horizon™ BV421  conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific Blue™ conjugates.

562564 Rev. 3
Format Details
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BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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BV421
Violet 405 nm
407 nm
423 nm
562564 Rev.3
Citations & References
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Development References (7)

  1. Bhattacharya A, Dorf ME, Springer TA. A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J Immunol. 1981; 127(6):2488-2495. (Immunogen: Immunoprecipitation). View Reference
  2. Ernst DN, McQuitty DN, Weigle WO, Hobbs MV. Expression of membrane activation antigens on murine B lymphocytes stimulated with lipopolysaccharide. Cell Immunol. 1988; 114(1):161-173. (Clone-specific: Flow cytometry). View Reference
  3. 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. (Clone-specific: Blocking). View Reference
  4. Hattori M, Buse JB, Jackson RA, et al. The NOD mouse: recessive diabetogenic gene in the major histocompatibility complex. Science. 1986; 231(4739):733-735. (Clone-specific). View Reference
  5. Nelson AJ, Hosier S, Brady W, Linsley PS, Farr AG. Medullary thymic epithelium expresses a ligand for CTLA4 in situ and in vitro. J Immunol. 1998; 151(5):2453-2461. (Clone-specific: Blocking, Immunofluorescence, Immunohistochemistry). View Reference
  6. Viville S, Neefjes J, Lotteau V, et al. Mice lacking the MHC class II-associated invariant chain. Cell. 1993; 72(4):635-648. (Clone-specific: Flow cytometry, Immunofluorescence). View Reference
  7. Yamashita I, Nagata T, Tada T, Nakayama T. CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection. Int Immunol. 1993; 5(9):1139-1150. (Clone-specific: Blocking). View Reference
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562564 Rev. 3

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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.