| Description |
The CD154 MicroBead Kit is an indirect magnetic labeling system for the enrichment of CD154+ antigen-specific CD4+ T cells from human PBMCs. CD154+ T cells are indirectly magnetically labeled with CD154-Biotin antibody and Anti-Biotin MicroBeads.
The CD154 antibody specifically recognizes the human 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. Due to its transient expression within hours after activation, CD154 can be used as a marker for activated antigen-specific CD4+ T cells1. Adding a CD40 blocking-antibody to the cell suspension during stimulation prevents down-regulation of CD154 expression induced by interaction with CD40 expressed on antigen-presenting cells. |
| Applications |
| The CD154 MicroBead Kit was developed for positive selection of activated CD154+ antigen-specific CD4+ T cells from human PBMCs. |
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| Figure 1 |
CD154+ T cells from PBMCs of a CMV+ donor were separated using the CD154 MicroBead Kit, two MS Columns, and a MiniMACS™ Separator.
PBMCs were stimulated for 16 hours with CMV pp65 – Recombinant Protein, and a CD40 blocking-antibody was added during the stimulation to prevent downregulation of CD154. Subsequently, CD154+ T cells were separated using the CD154 MicroBead Kit and stained with Anti-Biotin-PE and CD4-FITC. |
| CMV pp65–stimulated sample |
| A: Before separation |
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| B: Positive fraction |
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| Unstimulated control |
| C: Positive fraction |
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| D: Before separation |
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