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Purified Rat Anti-Mouse H2-M
Purified Rat Anti-Mouse H2-M

Two-color analysis of the cytoplasmic expression of H2-M in splenic B lymphocytes and dendritic cells. Fixed and permeabilized C57BL/6 splenocytes were stained with either purified rat IgG1, κ isotype control mAb R3-34 (Cat. No. 553922, top panels) or purified mAb 2E5A (bottom panels) in the presence of Mouse Fc Block™ (purified anti-mouse CD16/CD32 mAb 2.4 G2, Cat. No. 553141/553142), followed by FITC-conjugated anti-rat IgG1 mAb RG11/39.4 (Cat. No. 553892). B lymphocytes were identified by staining with PE-conjugated anti-mouse CD19 mAb 1D3 (Cat. No. 557399/553786, left panels), and dendritic cells were identified with PE-conjugated anti-mouse CD11c mAb HL3 (Cat. No. 557401/553802, right panels). Flow cytometry was performed on a BD FACSCalibur™ System (BD Biosciences, San Jose, CA).

Two-color analysis of the cytoplasmic expression of H2-M in splenic B lymphocytes and dendritic cells. Fixed and permeabilized C57BL/6 splenocytes were stained with either purified rat IgG1, κ isotype control mAb R3-34 (Cat. No. 553922, top panels) or purified mAb 2E5A (bottom panels) in the presence of Mouse Fc Block™ (purified anti-mouse CD16/CD32 mAb 2.4 G2, Cat. No. 553141/553142), followed by FITC-conjugated anti-rat IgG1 mAb RG11/39.4 (Cat. No. 553892). B lymphocytes were identified by staining with PE-conjugated anti-mouse CD19 mAb 1D3 (Cat. No. 557399/553786, left panels), and dendritic cells were identified with PE-conjugated anti-mouse CD11c mAb HL3 (Cat. No. 557401/553802, right panels). Flow cytometry was performed on a BD FACSCalibur™ System (BD Biosciences, San Jose, CA).

Product Details
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BD Pharmingen™
H2-DM
Mouse (QC Testing)
Rat IgG1, κ
Recombinant H2-DM protein
Intracellular staining (flow cytometry) (Routinely Tested), Immunoprecipitation (Reported)
0.5 mg/ml
AB_394380
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C.

Recommended Assay Procedures

For flow cytometry of leukocytes, it is recommended that Mouse Fc Block™ (purified anti-mouse CD16/CD32 mAb 2.4G2, Cat. No. 553141/553142) be used. If Mouse Fc Block™ is used, it is important that the second step anti-rat IgG antibody does not cross-react with the 2.4g2 mAb (rat IgG2b, κ);  we have found that FITC-labeled anti-rat IgG1 mAb RG11/39.4 (Cat. No. 553892) is effective.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  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. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
552405 Rev. 1
Antibody Details
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2E5A

H2-M, also known as H2-DM, is a non-classical MHC class II molecule in antigen-presenting cells. H2-M and CD74 (Ii) are critical components of the antigen-processing pathway of the classical MHC class II molecules. Like classical MHC class II molecules, non-classical MHC molecules have limited polymorphism. There are two isoforms of H2-M, αβ1 and αβ2, encoded by the H2-DMa gene and either H2-DMb1 or H2-DMb2, respectively. The 2E5A antibody reacts with H2-M αβ2 dimers (the predominant form transcribed in the mouse spleen), but not αβ1 dimers. H2-M dimers (both isoforms) are integral proteins of lysomal membranes, where they catalyze the release of CLIP (class II-associated Ii peptide) from the peptide-binding groove of classical MHC class II dimers and stabilize the open binding site to allow loading  of exogenous peptides. They also may facilitate the selection of high-affinity antigenic peptides by allowing the dissociation of poorly fitting non-CLIP peptides to be exchanged for higher affinity peptides. This peptide-exchange function of H2-M is essential for the intrathymic development of the repertoire of CD4+ T lymphocytes and the maturation of humoral and cell-mediated immune responses. H2-M is believed to be expressed in all cells which express classical MHC class II molecules, including peripheral B lymphocytes, macrophages, dendritic cells, thymic cortical epithelium, and some tumor cell lines. Its level of expression is at least partially controlled by CD74 (Ii), whereas its functional activity is negatively regulated by another non-classical MHC class II molecule, H2-O.

552405 Rev. 1
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
552405 Rev.1
Citations & References
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Development References (14)

  1. Alfonso C, Han JO, Williams GS, Karlsson L. The impact of H2-DM on humoral immune responses. J Immunol. 2001; 167(11):6348-6355. (Biology). View Reference
  2. Alfonso C, Karlsson L. Nonclassical MHC class II molecules. Annu Rev Immunol. 2000; 18:113-142. (Immunogen). View Reference
  3. Fung-Leung WP, Surh CD, Liljedahl M, et al. Antigen presentation and T cell development in H2-M-deficient mice. Science. 1996; 271(5253):1278-1281. (Biology). View Reference
  4. Honey K, Duff M, Beers C, et al. Cathepsin S regulates the expression of cathepsin L and the turnover of gamma-interferon-inducible lysosomal thiol reductase in B lymphocytes. J Biol Chem. 2001; 276(25):22573-22578. (Biology). View Reference
  5. Lang T, Ave P, Huerre M, Milon G, Antoine JC. Macrophage subsets harbouring Leishmania donovani in spleens of infected BALB/c mice: localization and characterization. Cell Microbiol. 2000; 2(5):415-430. (Biology). View Reference
  6. Lankar D, Vincent-Schneider H, Briken V, Yokozeki T, Raposo G, Bonnerot C. Dynamics of major histocompatibility complex class II compartments during B cell receptor-mediated cell activation. J Exp Med. 2002; 195(4):461-472. (Biology). View Reference
  7. Liljedahl M, Winqvist O, Surh CD, et al. Altered antigen presentation in mice lacking H2-O. Immunity. 1998; 8(2):233-243. (Biology). View Reference
  8. Pierre P, Shachar I, Matza D, Gatti E, Flavell RA, Mellman I. Invariant chain controls H2-M proteolysis in mouse splenocytes and dendritic cells. J Exp Med. 2000; 191(6):1057-1062. (Biology). View Reference
  9. Rodgers JR, Levitt JM, Cresswell P, et al. A nomenclature solution to mouse MHC confusion. J Immunol. 1999; 162(10):6294. (Biology). View Reference
  10. Tompkins SM, Padilla J, Dal Canto MC, Ting JP, Van Kaer L, Miller SD. De novo central nervous system processing of myelin antigen is required for the initiation of experimental autoimmune encephalomyelitis. J Immunol. 2002; 168(8):4173-4183. (Biology). View Reference
  11. Walter W, Lingnau K, Schmitt E, Loos M, Maeurer MJ. MHC class II antigen presentation pathway in murine tumours: tumour evasion from immunosurveillance. Br J Cancer. 2000; 83(9):1192-1201. (Biology). View Reference
  12. Walter W, Loos M, Maeurer MJ. Differential expression of alternative H2-M isoforms in B cells, dendritic cells and macrophages by proinflammatory cytokines. Mol Immunol. 1999; 36(11-12):733-743. (Biology). View Reference
  13. Walter W, Scheuer C, Lingnau K, et al. H2-M, a facilitator of MHC class II peptide loading, and its negative modulator H2-O are differentially expressed in response to proinflammatory cytokines. Immunogenetics. 2000; 51(10):794-804. (Biology). View Reference
  14. Walter W, Scheuer C, Loos M, Reichert TE, Maeurer MJ. H2-Mbeta 1 and H2-Mbeta 2 heterodimers equally promote clip removal in I-A(q) molecules from autoimmune-prone DBA/1 mice. J Biol Chem. 2001; 276(14):11086-11091. (Biology). View Reference
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552405 Rev. 1

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