Cannabinoid Receptor 2 (CB2) Receptor Internalization Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of CB2
• 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

Two different cannabinoid receptors named CB1 and CB2 have been characterized. Both of these receptors are Gi/0-coupled G protein-coupled receptors (GPCRs). CB1 and CB2 are activated by endocannabinoids such as anandamide. Moreover, several synthetic cannabinoids as well as receptor antagonists have been developed. The CB2 receptor is mainly expressed in cells and organs of the immune system and its activity is unrelated to the cannabinoid psychoactivity that is mediated by CB1. Like other GPCRs, the CB2 receptor is rapidly internalized following ligand binding.

Figure 1. Internalization of CB2-EGFP. Cells were treated with 0.25% DMSO (control, left panel), 10 nM WIN55,212-2 (center panel), or 10 nM WIN55,212-2 + 10 μM AM630 (right panel). Arrows indicate WIN55,212-2-induced CB2 internalization detected by the image analysis algorithm.

Figure 2. Concentration response curves in the CB2 agonist and antagonist assays. Concentration response was measured in 9 point half log dilution series. Cells were incubated with test compound for 2 hours in agonist mode and pre-incubated for 1 hour with test compound before incubation with WIN55,212-2 for 2 hours in antagonist mode. Cells were then fixed and internalization was measured by image analysis detecting cytoplasmic foci. A) WIN55,212-2 concentration response curve in the CB2 agonist Redistribution assay (n=8). The EC₅₀ value of WIN55,212-2 is approximately 3 nM. % activity was calculated relative to the positive (100 nM WIN55,212-2) and negative control (0.25% DMSO). B) AM630 concentration response curve in the CB2 antagonist Redistribution assay where 10 nM WIN55,212-2 is used as agonist (n=8). The EC₅₀ value of AM630 is approximately 180 nM. % activity was calculated relative to the positive (10 μM AM630) and negative control (0.25% DMSO).