CXCR4 Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of CXCR4
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The CXCR4 receptor is a chemokine receptor and belongs to the family of G protein coupled receptors (GPCRs). Its only known endogenous ligand is stromal cell-derived factor 1 (SDF-1), which exists in two forms originating from alternative splicing, i.e. SDF-1α and SDF-1β. SDF-1 and CXCR4 form an important chemokine ligand/receptor pair, that plays a crucial role in numerous biological processes including hematopoiesis, cardiogenesis, vasculogenesis, neuronal development, immune cell trafficking, malignant cell growth, and metastasis. Binding of SDF-1 to the CXCR4 receptor activates G-proteins and induces signaling through downstream pathways involving Ras and the PI3 kinase. CXCR4 signaling results in the activation of JAK2 and transcription factors such as AP-1 and STATs.

Figure 1. Internalization of CXCR4-EGFP. Cells were treated with 10 nM SDF-1α for 1 hr (right panel) or vehicle control (left panel). Arrows indicate the CXCR4 internalization detected by the image analysis algorithm.

Figure 2. Concentration response curves in the CXCR4 assay: A) SDF-1α concentration response in the CXCR4 agonist assay (n=8). The EC50 is approximately 3 nM. Concentration response was measured in 9 point half log dilution series. Cells were treated with SDF-1α for 1 hr. Cells were then fixed and receptor internalization was measured using the Cellomics ArrayScan® V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (10 nM SDF-1α) and negative control (0.25% DMSO). B) AMD3100 concentration response in the CXCR4 antagonist assay (n=8). The EC₅₀ is approximately 4 nM. Cells were treated with SDF-1α in the presence of a half log dilution series of AMD3100. Cells were then fixed and receptor internalization was measured using the Cellomics ArrayScan® V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (300 nM AMD3100) and negative control (0.25% DMSO)