CXCR4 Internalization – 2 Color Internalization and Trafficking Assay

CXCR4 (chemokine receptor 4) also known as fusin or CD184  is a G-Protein Coupled Receptor (GPCR) that is stimulated by stromal-derived-factor-1 (SDF1 also known as CXCL12).

The receptor is a co-receptor for HIV infection and has been implicated in many human diseases including AIDS, cancer and rheumatoid arthritis.  SpectraGenetics has developed a 2 color  assay that directly measures CXCR4 internalization, trafficking, and recycling back to the membrane using fluorogen activation peptide technology.

Fluorogen Activating Peptide (FAP) technology combines a genetic tag (to tag the target GPCR), and a fluorogenic dye that only gives signal when bound to the tag.

Using the FAP and cell-impermeant dyes you can selectively label cell-surface and endocytosed protein pools in the following wash-free protocol.


While the confocal images above confirm that the localization of the Green and Red signal to surface and endocytic pools respectively, the signal produced in the assay is homogeneous, so imaging is not required.  The plots below show the assay run on a BD FACS-Vantage cytometer, and are plots of the average fluorescence intensity from the red and green channels from 10,000 events per data point.

CXCR4 quantification

The left-hand panel shows a dose response of the receptor agonist CXCL12.  Increasing concentrations of agonist move the cells from predominantly surface GPCR  localization (labeled in Green after the color-exchange), to predominantly endocytosed GPCR (the Red fluorogen, protected from exchange within the endosome).

Similarly, the right-hand panel shows a time-course following agonist addition, demonstrating a smooth transition from mostly surface GPCR at T-zero, through a clear 50/50 point at around 40 minutes, to a predominantly endocytic localization after 80 minutes.

While it is possible to simply measure an increase in the Red channel to follow agonist-driven endocytosis, measuring the Green channel simultaneously allows you to take the Red/Green ratio or Green-Red difference to obtain a larger assay window.

The same cell line can also assay antagonist response or see internalized receptor return to the surface (re-sensitization) by following these steps:

  1. Add agonist plus red fluorogen and allow time for response
  2. Add green fluorogen
  3. Add test compound (antagonist) and allow time for response
  4. Read both red and green signal


The following products were used to obtain the data in this report:

Product Name



# Wells


βGREEN fluorogen


20 nmol



βRED fluorogen


20 nmol



CXCR4-FAPβ1- HEK293 Cell Line


2 vials



CXCR4-FAPβ1-U937 Cell Line


2 vials



SpectraGenetics has tagged more than 150 GPCRs and has validated more than 30 GPCR cell lines.

We also offer this assay as a service through our partner Sharp Edge Labs (

This report is available as a pdf.