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Multicolor flow cytometric analysis of I-A/I-E MHC Class II alloantigen expression on Mouse splenic leucocytes. BALB/c Mouse splenocytes 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 Hamster Anti-Mouse CD3e antibody (Cat. No. 553066/561826 and with either BD Horizon™ RY586 Rat IgG2b, κ Isotype Control (Cat. No. 568160; Left Plot) or BD Horizon™ RY586 Rat Anti-Mouse I-A/I-E antibody (Cat. No. 568545/568546 Right Plot) at 0.5 µg/test. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of I-A/I-E (or Ig Isotype control staining) versus CD3e was derived from gated events with the forward and side light-scatter characteristics of viable (DAPI-negative) 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.


BD Horizon™ RY586 Rat Anti-Mouse I-A/I-E

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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.
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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.

Development References (7)
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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
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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
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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
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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
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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
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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
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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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