CD154 ( #130-092-289 #130-092-290 #130-092-690 #130-096-217 #130-096-603 #130-096-793 #130-096-233 )

CD154

Overview

The antibody recognizes the human CD154 (CD40L) antigen and can be used to detect activated antigen-specific CD4⁺ T cells.
Suitable applications are

Identification and enumeration of activated CD154⁺ antigen-specific CD4⁺ T cells by flow cytometry or fluorescence microscopy.
Identification and enumeration of antigen-specific CD4⁺ T cells in combination with a MACS^® Cytokine Secretion Assay Kit.
Evaluation of intracellular cytokine expression in activated antigen-specific CD4⁺ T cells by using CD154 antibodies in combination with antibodies against human cytokines.

Details

Background information
The 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. The antibody has been shown to block the activation of antigen-presenting cells by T helper cells in vitro. Because of its transient expression within hours after activation, CD154 can be used as a marker for activated antigen-specific CD4+ T cells.1 The addition of a CD40-blocking antibody during the stimulation of cell suspensions prevents the down-regulation of CD154 expression induced by interaction with CD40 expressed on antigen-presenting cells. For intracellular detection of CD154 expression or if a pure population of enriched T cells is used, blocking of CD40 is not required.

Cross-reactivity

This monoclonal antibody was shown to cross-react with

Rhesus monkey (Macaca mulatta)
Cynomolgus monkey (Macaca fascicularis)

Clone	Isotype
5C8	Mouse IgG2a

Figure 1

Cell surface staining: Human PBMCs were restimulated in vitro with or without CMV pp65 – Recombinant Protein or CytoStim and CD40 antibody for 16 hours. Cells were harvested, stained with CD4-FITC or CD4-PE and CD154-PE (A, B), CD154-Biotin (C, D), CD154-VioBlue (E, F), or CD154-PE-Vio770 (G, H). Cells labeled with CD154-Biotin were stained with Anti-Biotin-PE in adition. Analysis was performed by flow cytometry, cell debris and dead cells were excluded from the analysis according to PI fluorescence.

A: CMV-pp65 stimulated sample

B: Unstimulated control

C: CMV pp65-stimulated sample

D: Unstimulated control

E: Cytostim stimulated sample

F: Unstimulated control

G: Stimulated sample

H: Unstimulated control

Figure 2

Intracellular staining: Human PBMCs were restimulated in vitro with or without SEB for 16 hours. Brefeldin A was added after 2 hours. The cells were harvested, stained with CD4-FITC, permeabilized, and intracellularly stained with CD154-APC-Vio770 and Anti-TNF-a-VioBlue (A, B). Cells were analyzed by flow cytometry. Gating was performed according to CD4 expression and side scatter properties. Cell debris and dead cells were excluded from the analysis using an FL-2 vs. FL-3 dot plot.

A: SEB stimulated sample

B: Unstimulated control

Figure 3

Rhesus monkey (A, B) and Cynomolgus monkey (C, D) PBMCs were stimulated using the T Cell Activation/Expansion Kit, non-human primate. B and D show unstimulated control samples. Cells were stained with CD154-PE and CD4-APC and analyzed by flow cytometry.