The CD154 MicroBead Kit has been developed for positive selection of activated CD154
+
antigen-specific CD4
+
T cells from human PBMCs.

Data and images for CD154 MicroBead Kit, human

Figures

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
View details

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.
View details

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.
After separation
CMV pp65–stimulated sample
Unstimulated control
View details

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.
View details

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.

Specifications for CD154 MicroBead Kit, human

Overview

The CD154 MicroBead Kit has been developed for positive selection of activated CD154
+
antigen-specific CD4
+
T cells from human PBMCs.

Detailed product information

Background information

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 cells
1
. 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.

Detailed separation procedure

CD154
+
T cells are indirectly magnetically labeled with CD154-Biotin antibody and Anti-Biotin MicroBeads UltraPure.

Columns

MS, LS, or autoMACS
®
Columns.

References for CD154 MicroBead Kit, human

Publications

  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

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