Description
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 cells1,2.
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.
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.
