Table 1.

Screening of ligand (X) activity in CHO-C5aR1/C3aR cells

Cells/ receptor	To screen for	Treatment scheme
CHO-C5aR1	Agonist	Directly stimulate cells with ligand X Positive control: hC5a (e.g., 100 nM)
	Antagonist	Pretreat cells with ligand X, then stimulate with 0.3 nM hC5a (∼EC80)
CHO-C3aR	Agonist	Directly stimulate cells with ligand X Positive control: hC3a (e.g., 100 nM)
	Antagonist	Pretreat cells with ligand X, then stimulate with 0.3 nM hC3a (∼EC80)
Counter-screening: Can similar effect be observed on other receptors?

Note: Human C5aR1 and C3aR demonstrate high degree of structural similarity and ligand promiscuity,¹³ as such, we recommend all potential hits for human C5aR1 to be counter-screened against human C3aR and vice versa. This test should be performed first prior to other more comprehensive selectivity screens.