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Protocol for cell-based screening assay to measure ERK1/2 phosphorylation as a readout for complement receptor
Xaria X Li1, Trent M Woodruff1
1School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia, QLD 4072 Australia.
STAR Protocols
|November 30, 2023
Summary
This study details a new in vitro protocol to screen complement receptor modulators. The method uses phospho-ERK as a readout for C3aR and C5aR1 activity in immune cells and cell lines.
Area of Science:
- Immunology
- Pharmacology
Background:
- Complement receptors C3aR and C5aR1 are key therapeutic targets.
- Understanding their modulation is crucial for developing new treatments.
Purpose of the Study:
- To present a detailed protocol for screening agonists and antagonists of C3aR and C5aR1 in vitro.
- To establish a reliable method using phosphorylated extracellular signal-regulated kinase (p-ERK) as a readout.
Main Methods:
- Isolation of human monocyte-derived macrophages.
- Culture and preparation of Chinese hamster ovary cells expressing human C3aR or C5aR1.
- Pharmacological assays and detection of p-ERK1/2 in cell lysates.
Main Results:
- The protocol enables effective screening of receptor modulators.
- Phospho-ERK1/2 levels serve as a quantifiable readout for receptor activity.
- The method is adaptable to various cell lines.
Conclusions:
- This protocol provides a robust platform for studying complement receptor pharmacology.
- It facilitates the discovery and characterization of novel therapeutic agents targeting C3aR and C5aR1.
