A Cellular Assay for the Identification of CXCR4-Interacting Agents

Published: February 29, 2024

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…

Disclosures

The authors have nothing to disclose.

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.

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Cite This Article
A Cellular Assay for the Identification of CXCR4-Interacting Agents. J. Vis. Exp. (Pending Publication), e21996, doi: (2024).

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