Background information

Detailed separation procedure

Columns

• Figure 1

  CD154 + T cells were isolated from human PBMCs of a CMV + donor using the CD154 MicroBead Kit, two MS Columns, and a MiniMACS™ Separator. PBMCs were stimulated for 16 hours with PepTivator CMV pp65 (# 130-093-438), a CD40 blocking-antibody was added during the stimulation to prevent down-regulation of CD154. Subsequently, CD154 + cells were separated using the CD154 MicroBead Kit. Cells were fluorescently stained with Anti-Biotin-PE (# 130-090-756) and CD4 (VIT4)-APC (# 130-092-374) to detect CD154 + cells. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

  Before separation

  CMV pp65–stimulated sample	Unstimulated control
  After separation

  CMV pp65–stimulated sample	Unstimulated control

• Selected references

  1. Frentsch, M. et al. (2005)
     Direct access to CD4

     ⁺

     T cells specific for defined antigens according to CD154 expression.
     Nat. Med. 11: 1118-1124
  2. Bacher, P. et al. (2014) Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi. Mucosal Immunol 7(4): 916-928
  3. Bacher, P. et al. (2013) Antigen-reactive T cell enrichment for direct, high-resolution analysis of the human naive and memory Tʜ cell repertoire. J. Immunol. 190(8): 3967-3976
  4. Schmied, S. et al. (2015)
     Analysis of the functional WT1-specific T-cell repertoire in healthy donors reveals a discrepancy between CD4

     ⁺

     and CD8

     ⁺

     memory formation.
     Immunology 145(4): 558-569

• Scientific posters

  Rapid antigen-reactive T cell enrichment procedure enablesdirect

  ex vivo

  BKV-specific CD4

  ⁺

  T cell characterization

• Customer reports and application notes

  Rapid antigen-reactive T cell enrichment (Rapid ARTE) - Workflow protocol
  Detection, enrichment, and direct phenotypic and functional characterization of antigen-specific naive and memory T cell subsets. Bacher, P. & Scheffold, A. (2015)