MRGPRX2-mediated Cellular Activation Assay

HTLA cells stably expressing MRGPRX2 (5×10⁴ cells/well) were plated into a 96-well plate with an antibiotic-free medium and incubated for 6 h to allow cells’ adherence at 37^°C with 5% CO₂. Following 6 h of incubation, the medium was aspirated, and cells were incubated with C9 (1 or 10 μM) in an antibiotic-free medium for 5 min, followed by stimulation with MRGPRX2 agonist for an additional 16 h at 37^°C with a 5% CO₂ incubator. After 16 h, the medium was aspirated and replaced with 100 μl of Bright-Glo solution (Promega). The relative luminescence unit was measured in a Thermo Labsystems Luminoskan Ascent 392 Microplate Luminometer (42 (link)). For HTLA cells transiently transfected with MRGPRX2 or its missense variant, V282M tango plasmid, cells treated with C9 (10 μM), C7 (10 μM), or non-treated control and assay were performed similarly (32 (link)).