Protocol for cell-based screening assay to measure ERK1/2 phosphorylation as a readout for complement receptor activation

Abstract

Summary: The complement receptors C3aR and C5aR1 are promising therapeutic targets. Here, we present a protocol to screen the effects of different agonists and antagonists on these receptors in vitro, using phosphorylated extracellular signal-regulated kinase (ERK) as a readout. We describe steps for isolating human monocyte-derived macrophages, culturing and preparing Chinese hamster ovary cells stably expressing human C5aR1 or C3aR, performing pharmacological assays, and detecting phospho-ERK1/2 in the cell lysate. This protocol can also be performed using other cell lines.For complete details on the use and execution of this protocol, please refer to Li et al. (2020)1 and Li et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell culture
• Cell-based Assays
• High-Throughput Screening
• Immunology