C3AR1 Knockout THP-1 Cell Line

The C3AR1 Knockout THP-1 Cell Line is a CRISPR/Cas9-edited knockout cell line derived from the human monocytic THP-1 cell line, engineered for loss-of-function studies of the complement component C3a receptor (C3AR1). This product provides a stable, gene-disrupted model in which C3AR1 expression is ablated, enabling researchers to dissect C3a-mediated signaling in a macrophage-relevant cellular context. The cell line retains the parental THP-1 background and can be maintained in suspension or differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA) or cytokines, preserving utility while eliminating C3a responsiveness.

The THP-1 host cell line was established from peripheral blood of a one-year-old male with acute monocytic leukemia and is widely used for studying monocyte/macrophage differentiation and function. These non-adherent suspension cells can be differentiated into adherent, macrophage-like cells with PMA or cytokines such as GM-CSF or IFN-??, exhibiting phagocytosis, cytokine secretion, and pattern recognition receptor expression. This knockout derivative allows direct interrogation of C3AR1-specific roles in macrophage biology without variable receptor expression.

C3AR1 is a G protein-coupled receptor for the anaphylatoxin C3a, generated during complement activation via classical, lectin, or alternative pathways. Ligand binding triggers G??i/o-dependent PLC?? activation, leading to IP3-mediated calcium release and PKC activation. Concurrent MAP kinase signaling results in ERK and p38 phosphorylation, which drive NF-??B-dependent transcription of pro-inflammatory cytokines (IL-6, IL-8, TNF-??) and reactive oxygen species. ??-arrestin recruitment and potential crosstalk with TLR pathways further amplify inflammation. Upstream, C3AR1 activity is regulated by C3 convertase activity and cytokines such as TNF-?? and IL-1??, which modulate receptor expression.

In the THP-1 model, C3a stimulation triggers chemotaxis, degranulation, and robust cytokine release??responses abolished in C3AR1 knockout cells, confirming the receptor??s essential role in complement-mediated macrophage activation. This disruption provides a clean background to map C3AR1-dependent signaling and distinguish it from related receptors like C5aR1. The knockout line is particularly valuable for validating pharmacological targets in complement-driven inflammatory pathologies and assessing novel C3AR1 antagonist selectivity. Moreover, it facilitates elucidation of C3AR1-TLR crosstalk, as THP-1 cells express a repertoire of immune sensors that cooperate with complement signals to shape cytokine profiles.

Typical applications include measuring C3a-induced calcium flux in fura-2?Cloaded cells, transwell chemotaxis assays toward C3a gradients, and ELISA-based quantification of IL-6, IL-8, and TNF-?? secretion. Western blot analysis of phospho-ERK and phospho-p38 offers additional confirmation, while RT-qPCR panels for NF-??B?Cdependent genes provide transcriptional readouts. Flow cytometry can verify C3AR1 surface expression loss. For disease modeling, the knockout line supports studies of sepsis, rheumatoid arthritis, ischemia-reperfusion injury, and allergic asthma, particularly in co-culture or inflammasome-activating conditions. Detailed protocols and quality control data are available upon request. For further information, contact Ascent Research.