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).