Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays.

Area of Science:

• Pharmacology
• Biotechnology
• Cell Biology

Background:

• Label-free impedance assays are valuable for studying G protein-coupled receptor (GPCR) activation in real-time.
• Current methods face limitations in throughput and cost for large-scale experiments like dose-response studies.
• High-throughput screening requires efficient and cost-effective assay methodologies.

Purpose of the Study:

• To develop and validate a serial agonist addition protocol to improve the efficiency of label-free GPCR assays.
• To enable the establishment of complete dose-response curves from a single cell culture well.
• To overcome the limitations of sequential well-by-well recordings in high-throughput settings.

Main Methods:

• A serial agonist addition protocol was developed for label-free impedance-based assays.
• Increasing concentrations of GPCR agonists were sequentially added to a single cell layer.
• Impedance was continuously monitored throughout the agonist addition process (agonist mode).
• The protocol was also adapted to assess receptor blocking by antagonists (antagonist mode).

Main Results:

• A full dose-response curve for a GPCR agonist can be generated from a single cell layer using the serial addition method.
• The protocol is effective across different GPCR coupling types (Gq, Gi/o, Gs).
• The method is compatible with both recombinant and endogenous receptor expression levels.

Conclusions:

• The serial agonist addition protocol significantly increases the output and efficiency of label-free GPCR impedance assays.
• This approach reduces the cost associated with disposable electrode arrays and time constraints of sequential recordings.
• The protocol offers a versatile and robust method for studying GPCR activation and antagonist effects.