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Purified NA/LE Hamster Anti-Mouse CD154
Purified NA/LE Hamster Anti-Mouse CD154

CD154 expression on activated and resting T lymphocytes. BALB/c spleen T cells, purified on a T Cell Enrichment Column (R&D Systems), were cultured for 8 hours in the presence (Panels A and C) or absence (Panels B and D) of plate-bound 500A2 antibody (anti-CD3e, Cat. No. 553238). They were stained with NA/LE™ MR1 antibody (Panels A and B), followed by biotinylated mouse anti-hamster IgG, Cat. No. 554010, then Streptavidin-PE, Cat. No. 554061, (Panels A, B, C, and D). Flow cytometry was performed on a BD FACScan™ Flow Cytometry System.

CD154 expression on activated and resting T lymphocytes. BALB/c spleen T cells, purified on a T Cell Enrichment Column (R&D Systems), were cultured for 8 hours in the presence (Panels A and C) or absence (Panels B and D) of plate-bound 500A2 antibody (anti-CD3e, Cat. No. 553238). They were stained with NA/LE™ MR1 antibody (Panels A and B), followed by biotinylated mouse anti-hamster IgG, Cat. No. 554010, then Streptavidin-PE, Cat. No. 554061, (Panels A, B, C, and D). Flow cytometry was performed on a BD FACScan™ Flow Cytometry System.

Product Details
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BD Pharmingen™
CD40 Ligand; CD40L; gp39; Ly-62; Tnfsf5; T-BAM; HIGM1; IMD3
Mouse (QC Testing)
Armenian Hamster IgG3, κ
Activated mouse Th1 clone D1.6
Flow cytometry (Routinely Tested), Blocking, Immunohistochemistry (Reported)
1.0 mg/ml
21947
AB_394975
No azide/low endotoxin: Aqueous buffered solution containing no preservative, 0.2µm sterile filtered. Endotoxin level is ≤0.01 EU/µg (≤0.001 ng/µg) of protein as determined by the LAL assay.
RUO


Preparation And Storage

Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. This preparation contains no preservatives, thus it should be handled under aseptic conditions.

Recommended Assay Procedures

Flow cytometry:  For the detection of mouse CD154 on activated peripheral T cells, it is strongly recommended that T cells be purified before activation. Mouse CD154 is transiently expressed on the surfaces of activated normal T cells and certain T cell clones with a maximal level detected 6-8 hours post-activation. Activation with immobilized anti-CD3e mAb (e.g., 145-2C11, Cat. No. 557306/553058, or 500A2, Cat. No. 553238) is sufficient to induce CD154 expression on CD4+ cells. It has been reported that CD8+ cells express CD154 only in response to PMA/Ionomycin treatment. Therefore, for detection of CD154, it is crucial to utilize the proper activation stimuli and to stain cells at the optimal time for CD154 expression. We recommend the use of biotinylated mouse anti-hamster IgG cocktail (Cat. No. 554010) followed by a "bright" second-step reagent, such as Streptavidin-PE (Cat. No. 554061), for optimal detection of CD154.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Although hamster immunoglobulin isotypes have not been well defined, BD Biosciences Pharmingen has grouped Armenian and Syrian hamster IgG monoclonal antibodies according to their reactivity with a panel of mouse anti-hamster IgG mAbs. A table of the hamster IgG groups, Reactivity of Mouse Anti-Hamster Ig mAbs, may be viewed at http://www.bdbiosciences.com/documents/hamster_chart_11x17.pdf.
  3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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Antibody Details
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MR1

The MR1 monoclonal antibody specifically binds to CD154 (CD40 Ligand, gp39), an accessory molecule expressed on activated T helper (CD4+) lymphocytes. CD154 has also been detected on other types of leukocytes, including CD8+ T cells, medullary thymocytes, activated CD4+ NK-T cells, and human NK cells. CD154 plays an important role in costimulatory interactions between T and B lymphocytes and between antigen-presenting cells and lymphocytes, regulating the immune response at multiple levels. MR1 mAb inhibits in vitro activation of B lymphocytes by T helper cells by blocking interaction of gp39 with CD40. In vitro interactions of T cells and antigen-presenting cells can also be blocked by the MR1 antibody. In vivo treatment with MR1 antibody blocks the development of experimental autoimmune disease, inhibits formation of germinal centers and generation of memory B cells, reduces T-lymphocyte responses to allogeneic cells and allografts, prevents intrathymic deletion of self-reactive T lymphocytes, and disrupts antigen-specific T-cell responses.

553655 Rev. 15
Format Details
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NA/LE
NA/LE refers to the culture and purification methods and buffer used to produce purified antibodies with no azide and low endotoxin: Aqueous buffered solution containing no preservative, 0.2µm sterile filtered. Endotoxin level is ≤0.01 EU/µg (≤0.001 ng/µg) of protein as determined by the LAL assay.NA/LE are perfectly suited to be used in culture or in vivo (for nonhuman studies) for functional assays — blocking, neutralizing, activation or depletion — where the presence of azide may damage cells or exogenous endotoxin may signal or activate cells.
NA/LE
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Citations & References
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Development References (24)

  1. Carbone E, Ruggiero G, Terrazzano G, et al. A new mechanism of NK cell cytotoxicity activation: the CD40-CD40 ligand interaction. J Exp Med. 1997; 185(12):2053-2060. (Biology). View Reference
  2. DeKruyff RH, Gieni RS, Umetsu DT. Antigen-driven but not lipopolysaccharide-driven IL-12 production in macrophages requires triggering of CD40. J Immunol. 1997; 158(1):359-366. (Clone-specific: Blocking). View Reference
  3. Dunn RJ, Luedecker CJ, Haugen HS, Clegg CH, Farr AG. Thymic overexpression of CD40 ligand disrupts normal thymic epithelial organization. J Histochem Cytochem. 1997; 45(1):129-141. (Clone-specific: Immunohistochemistry). View Reference
  4. Durie FH, Fava RA, Foy TM, Aruffo A, Ledbetter JA, Noelle RJ. Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science. 1993; 261(5126):1328-1330. (Clone-specific: Blocking). View Reference
  5. Foy TM, Laman JD, Ledbetter JA, Aruffo A, Claassen E, Noelle RJ. gp39-CD40 interactions are essential for germinal center formation and the development of B cell memory. J Exp Med. 1994; 180(1):157-163. (Clone-specific: Blocking). View Reference
  6. Foy TM, Page DM, Waldschmidt TJ, et al. An essential role for gp39, the ligand for CD40, in thymic selection. J Exp Med. 1995; 182(5):1377-1388. (Clone-specific: Blocking). View Reference
  7. Garside P, Ingulli E, Merica RR, Johnson JG, Noelle RJ, Jenkins MK. Visualization of specific B and T lymphocyte interactions in the lymph node. Science. 1998; 281(5373):96-99. (Clone-specific: Blocking). View Reference
  8. Graca L, Honey K, Adams E, Cobbold SP, Waldmann H. Cutting edge: anti-CD154 therapeutic antibodies induce infectious transplantation tolerance. J Immunol. 2000; 165(9):4783-4786. (Clone-specific: Blocking). View Reference
  9. Grewal IS, Flavell RA. CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol. 1998; 16:111-135. (Biology). View Reference
  10. Griggs ND, Agersborg SS, Noelle RJ, Ledbetter JA, Linsley PS, Tung KS. The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of self-reactive T cells. J Exp Med. 1996; 183(3):801-810. (Clone-specific: Blocking). View Reference
  11. Kalled SL, Cutler AH, Datta SK, Thomas DW. Anti-CD40 ligand antibody treatment of SNF1 mice with established nephritis: preservation of kidney function. J Immunol. 1998; 160(5):2158-2165. (Clone-specific: Blocking). View Reference
  12. Kawano T, Cui J, Koezuka Y, et al. CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science. 1997; 278(5343):1626-1629. (Clone-specific: Blocking). View Reference
  13. Kelsall BL, Stuber E, Neurath M, Strober W. Interleukin-12 production by dendritic cells. The role of CD40-CD40L interactions in Th1 T-cell responses. Ann N Y Acad Sci. 1996; 795:116-126. (Clone-specific: Blocking). View Reference
  14. Laman JD, Claassen E, Noelle RJ. Functions of CD40 and its ligand, gp39 (CD40L). Crit Rev Immunol. 1996; 16(1):59-108. (Biology). View Reference
  15. Larsen CP, Elwood ET, Alexander DZ, et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature. 1996; 381(6581):434-438. (Clone-specific: Blocking). View Reference
  16. Lettesjö H, Burd GP, Mageed RA. CD4+ T lymphocytes with constitutive CD40 ligand in preautoimmune (NZB x NZW)F1 lupus-prone mice: phenotype and possible role in autoreactivity. J Immunol. 2000; 165(7):4095-4104. (Clone-specific: Immunohistochemistry). View Reference
  17. Masten BJ, Yates JL, Pollard Koga AM, Lipscomb MF. Characterization of accessory molecules in murine lung dendritic cell function: roles for CD80, CD86, CD54, and CD40L. Am J Respir Cell Mol Biol. 1997; 16(3):335-342. (Clone-specific: Blocking). View Reference
  18. Miga AJ, Masters SR, Durell BG, et al. Dendritic cell longevity and T cell persistence is controlled by CD154-CD40 interactions. Eur J Immunol. 2001; 31(3):959-965. (Clone-specific: Blocking). View Reference
  19. Nishimura T, Kitamura H, Iwakabe K, et al. The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes. Int Immunol. 2000; 12(7):987-994. (Clone-specific: Blocking). View Reference
  20. Noelle RJ, Roy M, Shepherd DM, Stamenkovic I, Ledbetter JA, Aruffo A. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc Natl Acad Sci U S A. 1992; 89(14):6550-6554. (Immunogen: Blocking). View Reference
  21. Roy M, Aruffo A, Ledbetter J, Linsley P, Kehry M, Noelle R. Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune responses. Eur J Immunol. 1995; 25(2):596-603. (Clone-specific: Blocking). View Reference
  22. Roy M, Waldschmidt T, Aruffo A, Ledbetter JA, Noelle RJ. The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells. J Immunol. 1993; 151(5):2497-2510. (Biology). View Reference
  23. Tian L, Noelle RJ, Lawrence DA. Activated T cells enhance nitric oxide production by murine splenic macrophages through gp39 and LFA-1. Eur J Immunol. 1995; 25(1):306-309. (Clone-specific: Blocking). View Reference
  24. Tomura M, Yu WG, Ahn HJ, et al. A novel function of Valpha14+CD4+NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system. J Immunol. 1999; 163(1):93-101. (Biology). View Reference
View All (24) View Less
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