Competitive Binding Assay to Identify Compounds Disrupting Receptor-Ligand Interactions

Abstract

Source: Schoofs, G., et al. A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4. J. Vis. Exp. (2018).

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

1. Prepare a stock solution of CXCL12^AF647 (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.
2. Prepare assay buffer by adding 40 mL HEPES (1 M, 20 mM final concentration) to 200 mL Hank's Balanced Salt Solution (HBSS, 10x, without phenol red and without sodium bicarbonate, 1x f....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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<br/> bottom, clear	Thermo Scientific	611U96
Bovine Serum Albumin (BSA)	Sigma	A1933-25G
HBSS (10x), calcium, magnesium, no phenol red	Gibco (Life Technologies)	14065-049
HEPES (1M)	Gibco (Life Technologies)	15630-056
Dulbecco's Phosphate Buffered Saline (DPBS)	Gibco (Life Technologies)	14190-094
Jurkat cells	ATCC
Reagent reservoir PP	Sigma	BR703411
Falcon tubes, 50ml	Greiner Bio-One	227 261