A Cellular Assay for the Identification of CXCR4-Interacting Agents

Abstract

Source: Claes, S. et al., A Kinetic Fluorescence-based Ca²⁺ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators. J. Vis. Exp. (2018)

This video illustrates a cellular assay involving glioblastoma cells expressing CXCR4 chemokine receptors. The assay employs a calcium-sensitive dye to measure fluorescence changes upon agent interaction, offering insights into cellular signaling dynamics.

Protocol

NOTE: All steps described under sections 1 and 2 are carried out under sterile conditions in a laminar flow cabinet. 1. Maintenance of U87.CD4.hCXCR4 Cells Grow the cells in T75 culture flasks at 37°C and 5% CO2 in a humidified incubator. NOTE: The in vitro cell line used in this protocol is a U87 glioblastoma cell line stably expressing cluster of differentiation 4 (CD4) and human CXC…

Materials

Fluo-2 AM	Abcam	ab142775	Fluorescent Ca2+ sensitive dye
Pluronic F-127	Sigma	P2443-250G	Pluronic acid
Gelatin	Sigma	G9391
AMD3100	Sigma	A5602-5 mg	Specific CXCR4 antagonist
Maraviroc	kind gift of AnorMed		Antiretroviral drug, CCR5 antagonist
Chemokine ligand CXCL12	PeproTech	300-28A
Chemokine ligand CCL5	PeproTech	300-06
Fetal Bovine Serum (FBS)	Gibco (Life Technologies)	10270-106
Bovine Serum Albumin (BSA)	Sigma	A1933-25G
Dulbecco's Modified Eagle's Medium (DMEM)	Gibco (Life Technologies)	41965-039
HBSS (10 x), calcium, magnesium, no phenol red	Gibco (Life Technologies)	14065-049
HEPES (1 M)	Gibco (Life Technologies)	15630-056
Trypsin-EDTA (0,25 %), phenol red	Gibco (Life Technologies)	25200-056
Dulbecco's Phosphate Buffered Saline (DPBS)	Gibco (Life Technologies)	14190-094
Falcon tubes, 50 mL	Greiner Bio-One	227 261
Tissue culture flask (T75)	Corning	353024
Black plate, 96-well, clear bottom, with lid	Costar/Fisher Scientific	10530753	Assay plate (96-well), for cell seeding
Polypropylene (PP) plates	Thermo Scientific (VWR)	732-2661	Plates used to prepare the compound plates and chemokine plates, round bottom
FLIPR Tetra high throughput cellular screening system	Molecular Devices		Fluorescent plate reader with integrated pipettor head and ICCD camera
FLIPR Tetra LED Module 470 – 495 nm	Molecular Devices	0200-6128	Light emitting diodes for excitation of the fluorescent Ca2+ sensitive dye
FLIPR Tetra Emission Filter 515 – 575 nm	Molecular Devices	0200-6203	Emission filter compatible with the fluorescent dye
FLIPR Tetra 96 Head	Molecular Devices	0310-4536	96-well pipettor head, integrated within the fluorescent plate reader
ScreenWorks	Molecular Devices		Software package used for data analysis and visualization on the FLIPR Tetra
Vi-CELL	Beckman Coulter		Cell viability analyzer
Corning CellBIND 96 Well Flat Clear Bottom Black Polystyrene Microplates, with Lid, Sterile	Corning	3340	Pre-coated 96-well assay plates that may represent an alternative for manual coating of the assay plate.