HTLA cells expressing either WT MRGPRX2 or its variants (5 × 104 cells per well) were plated into a 96-well plate in triplicates in a total volume of 160 μL antibiotic-free medium and incubated for 6 h at 37 °C to allow attachment. After 6 h, the medium was aspirated, and cells were incubated with MRGPRX2 ligands in 160 μL antibiotic-free medium for additional 16 h at 37 °C. The medium and ligands were then aspirated and 100 μL of Bright-Glo solution (Promega) was added to each well. Relative luminescence unit was measured in a Thermo Labsystems Luminoskan Ascent 392 Microplate Luminometer [20 (link),25 (link)].
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