384 well flat bottomed plates

HEK293 cells obtained from the JCRB Cell Bank (Osaka, Japan) were transiently transfected with MRGPRX2 and its mutants. Lipofectamine 2000 Reagent (Thermo Fisher Scientific Inc., Waltham, MA) and pcDNA3.1(+) containing each gene were diluted and mixed using Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific Inc.) to prepare lipid-DNA complexes (final concentrations: lipofectamine 2.5 µL/mL and DNA 2500 ng/mL). HEK293 cells were detached using TrypLE Express (Thermo Fisher Scientific Inc.) and prepared to 7 × 10⁵ cells/mL with the lipid-DNA complex. Thereafter, 25 µL of cells (1.75 × 10⁴ cells/well) were seeded per well in 384-well flat-bottomed plates (Corning Incorporated, Corning, NY) and incubated overnight at 37 °C under 5% CO₂ conditions. Cells treated with plasmid-free lipid solution were used as a negative control (untransfected cells).

HEK293 cells obtained from the JCRB Cell Bank (Osaka, Japan) were transfected transiently with dog MRGPRD, MRGPRF, MRGPRG, or MRGPRX2 or human MRGPRX2. Lipofectamine 2000 Reagent (Thermo Fisher Scientific Inc.) and pcDNA3.1(+) vector containing each gene were diluted and mixed using Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific Inc.) to prepare lipid-DNA complexes (final concentrations: lipofectamine 2.5 μL/mL and DNA 2,500 ng/mL). HEK293 cells were detached using TrypLE™ Express (Thermo Fisher Scientific Inc.) and prepared to 7 × 10⁵ cells/mL with the lipid-DNA complex. Thereafter, 25 μL cells (1.75 × 10⁴ cells/well) were seeded per well in 384-well flat-bottomed plates (Corning Incorporated, Corning, NY, USA) and incubated overnight at 37 °C under 5% CO₂ conditions. Cells treated with plasmid-free lipid solution were used as negative control (non-transfected cells).