CD154 Enrichment and Detection Kit ( #130-093-129 )

CD154 Enrichment and Detection Kit

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. Cell stimulation and fluorescent labeling for subsequent cell detection and enrichment are conveniently performed in a single step.

Details

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 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.

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. 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

A: Before separation

B: After separation

Unstimulated control

C: Before separation

D: After separation