CD154
Description Clone MR1 specifically recognizes the mouse CD154 antigen, a 39 kDa transmembrane glycoprotein also known as CD40L, gp39, T-BAM, TRAP, or Ly-62. CD154 is transiently up-regulated on activated CD4+ T cells and plays an important role as a costimulatory molecule in T cell/antigen-presenting cell interactions through ligation of CD40. The antibody has been shown to block the activation of antigen-presenting cells by T helper cells in vitro. Due to its transient expression within hours after activation, CD154 can be used as a marker for activated antigen-specific CD4+ T cells. Adding a CD40-blocking antibody during the stimulation of cell suspensions prevents the down-regulation of CD154. Blocking of CD40 is not required for intracellular detection of CD154 expression or if a pure population of enriched T cells is used. Applications The CD154 antibodies are suitable to identify and enumerate activated antigen-specific CD4+ T cells by flow cytometry or fluorescence microscopy, analysis of antigen-specific CD4+ T cells in combination with a MACS® Mouse Cytokine Secretion Assay – Detection Kit, or evaluation of intracellular cytokine expression in activated antigen-specific CD4+ T cells by using CD154 antibodies in combination with antibodies against mouse cytokines. Clone Isotype MR1 Hamster IgG3		Figure 1 Splenocytes from a KLH-immunized mouse were restimulated in vitro with or without KLH, CD28, and CD40 antibodies for six hours. Cells were harvested, stained with CD4-FITC and CD154-PE, or CD154-Biotin and Anti-Biotin-PE, and analyzed by flow cytometry. A lymphocyte gate based on forward and side scatter properties was activated. Dead cells and B cells were excluded according to PI and CD45R/B220-PerCP™ staining. A: KLH-stimulated sample B: Unstimulated control C: KLH-stimulated sample D: Unstimulated control

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