In this video, we describe a flow cytometry-based competitive binding assay to detect the interactions between the CXC Chemokine Receptor 4 (CXCR4) and its fluorescently-labeled natural ligand CXC Chemokine Ligand 12 (CXCL12), in the presence of a CXCR4-targeting small molecule. Incubating CXCR4-expressing cells with lower small molecule concentrations allows CXCR4-CXCL12 binding to some extent, which progressively declines upon a gradual increase in small molecule concentrations.
Protocol
1. Preparation of CXCL12, Assay Buffer, and Jurkat Cells for the Competition Binding Assay Prepare a stock solution of CXCL12AF647 (20 µg/mL; see Table of Materials and Reagents) by dissolving the lyophilized reagent (stored at -80 °C, in the dark) in ultrapure water supplemented with 0.01% (volume/volume) of Polysorbate 20. Store single-use aliquots from this stock solution at -80 °C, protected from light. Prepare assay buffer by adding 40…
Representative Results
Figure 1: Overview of the workflow and illustration of the type of data obtained. (A). The main steps of the protocol are schematized for the negative and positive control samples, and compound-treated samples. Samples without compound pre-incubation and without addition of fluorescently labeled chemokine (CXCL12AF647) are included to determine the background (auto)flu…
Disclosures
The authors have nothing to disclose.
Materials
BD FACSCanto II
Becton Dickinson
Not applicable
Flow cytometry device
BD FACSDIVA Software
BD FACSArray
Becton Dickinson
Not applicable
Flow cytometry device
BD FACSArray System Software
Rapid flow filter: 0.2 μm aPES
Thermo Scientific
566-0020
FlowJo
FlowJo is now a wholly owned subsidiary of BD.
Vi-CELL
Beckman Coulter
Not applicable
Cell viability analyzer
Sigma 3-18 KS
Sigma
Not applicable
Centrifuge
AMD3100
Sigma
A5602-5mg
Specific CXCR4 antagonist
h-SDF1a (AF647)
ALMAC
CAF-11-B-01
Fluorescently-labeled CXCL12, CXCL12AF647
Sterilin microtiter plate, 96-well, U
bottom, clear