The CD154 Enrichment and Detection Kit is designed for the easy identification and enrichment of activated antigen-specific CD4+ T cells from single-cell suspensions derived from mouse lymphoid tissues. The used Anti-PE MicroBeads UltraPure enhances sensitivity and minimizes background of debris and dead cells. Cell stimulation and fluorescent labeling for subsequent cell detection and enrichment are conveniently performed in a single step.

Data and images for CD154 Enrichment and Detection Kit (PE), mouse

Figures

Figure 1

Antigen-specific CD154
+
T cells were enriched using the CD154 Enrichment and Detection Kit, two MS Columns, and a MiniMACS™ Separator. Spleen cells from a KLH immunized BALB/c mouse were restimulated in vitro with 100 μg/mL KLH or were not restimulated in the presence of the CD154 Detection Cocktail (PE) for 6 hours.
CD154
+
T cells were magnetically isolated using Anti-PE MicroBeads UltraPure. Cells were fluorescently stained with CD4-FITC and CD45R (B220)-PerCP. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence. CD45R
+
cells were excluded from the analysis.
KLH-stimulated sample
Unseparated fraction
After separation
View details

Figure 1

Antigen-specific CD154
+
T cells were enriched using the CD154 Enrichment and Detection Kit, two MS Columns, and a MiniMACS™ Separator. Spleen cells from a KLH immunized BALB/c mouse were restimulated in vitro with 100 μg/mL KLH or were not restimulated in the presence of the CD154 Detection Cocktail (PE) for 6 hours.
CD154
+
T cells were magnetically isolated using Anti-PE MicroBeads UltraPure. Cells were fluorescently stained with CD4-FITC and CD45R (B220)-PerCP. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence. CD45R
+
cells were excluded from the analysis.
View details

Figure 1

Antigen-specific CD154
+
T cells were enriched using the CD154 Enrichment and Detection Kit, two MS Columns, and a MiniMACS™ Separator. Spleen cells from a KLH immunized BALB/c mouse were restimulated in vitro with 100 μg/mL KLH or were not restimulated in the presence of the CD154 Detection Cocktail (PE) for 6 hours.
CD154
+
T cells were magnetically isolated using Anti-PE MicroBeads UltraPure. Cells were fluorescently stained with CD4-FITC and CD45R (B220)-PerCP. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence. CD45R
+
cells were excluded from the analysis.
Unstimulated control
Unseparated fraction
After separation
View details

Figure 1

Antigen-specific CD154
+
T cells were enriched using the CD154 Enrichment and Detection Kit, two MS Columns, and a MiniMACS™ Separator. Spleen cells from a KLH immunized BALB/c mouse were restimulated in vitro with 100 μg/mL KLH or were not restimulated in the presence of the CD154 Detection Cocktail (PE) for 6 hours.
CD154
+
T cells were magnetically isolated using Anti-PE MicroBeads UltraPure. Cells were fluorescently stained with CD4-FITC and CD45R (B220)-PerCP. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence. CD45R
+
cells were excluded from the analysis.
View details

Figure 1

Antigen-specific CD154
+
T cells were enriched using the CD154 Enrichment and Detection Kit, two MS Columns, and a MiniMACS™ Separator. Spleen cells from a KLH immunized BALB/c mouse were restimulated in vitro with 100 μg/mL KLH or were not restimulated in the presence of the CD154 Detection Cocktail (PE) for 6 hours.
CD154
+
T cells were magnetically isolated using Anti-PE MicroBeads UltraPure. Cells were fluorescently stained with CD4-FITC and CD45R (B220)-PerCP. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence. CD45R
+
cells were excluded from the analysis.

Specifications for CD154 Enrichment and Detection Kit (PE), mouse

Overview

The CD154 Enrichment and Detection Kit is designed for the easy identification and enrichment of activated antigen-specific CD4+ T cells from single-cell suspensions derived from mouse lymphoid tissues. The used Anti-PE MicroBeads UltraPure enhances sensitivity and minimizes background of debris and dead cells. Cell stimulation and fluorescent labeling for subsequent cell detection and enrichment are conveniently performed in a single step.

Detailed product information

Background information

CD154, also known as CD40L, gp39, T-BAM, and TRAP is transiently up-regulated on activated CD4
+
T cells and plays an important role as a costimulatory molecule in the interaction between T cells and antigen-presenting cells through ligation of CD40. Due to its transient expression within hours of activation, CD154 can be used as a marker for activated antigen-specific CD4
+
T cells
1,2
.

Detailed procedure

Cells are activated with a desired antigen to induce CD154 expression and simultaneously incubated with the CD154 Detection Cocktail (PE) included in the CD154 Enrichment and Detection Kit. For optimal detection of CD154 on antigen-specific CD4
+
T cells, the CD154 Detection Cocktail contains i) CD154-PE, ii) a CD40 blocking antibody to prevent the down-regulation of CD154 surface expression, and iii) a CD28 antibody to provide a costimulatory signal. After a short incubation period, cells are magnetically labeled with Anti-PE MicroBeads UltraPure and separated over a MACS Column. The enriched antigen-specific CD154
+
T cells are eluted as the positive fraction and subjected to flow cytometric analysis.

Columns

MS, LS, or autoMACS
®
Columns.

Resources for CD154 Enrichment and Detection Kit (PE), mouse

References for CD154 Enrichment and Detection Kit (PE), mouse

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. Chattopadhyay, P. K. et al. (2005)
    A live-cell assay to detect antigen-specific CD4
    +
    T cells with diverse cytokine profiles.
    Nat Med 11: 1113-1117
  3. Kirchhoff, D. et al. (2007) Identification and isolation of murine antigen-reactive T cells according to CD154 expression. Eur. J. Immunol. 37: 2370-2377
  4. Rausch, S. et al. (2008) Functional analysis of effector and regulatory T cells in a parasitic nematode infection. Infect. Immun. 76(5): 1908-1919

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