Detection of intracellular calcium increase

We developed a new method to measure intracellular calcium change using NanoBiT technology [58]. HEK293 cells stably expressing Gα_qi chimera were seeded in 96-well plates at a cell density of 2×10⁴ cells/well. The next day, 30 ng of receptor plasmids, 30 ng of plasmids containing calmodulin tagged with SmBiT at C-terminal, and 30 ng of plasmids containing MYLK2S fused to LgBiT at the N-terminal were mixed with 0.2 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and added to the plated cells. Following transfection, steps were performed according to the manufacturer’s instructions. After 24 h, media was replaced with 100 μl of Opti-MEM, and cells were stabilized for 10 min at room temperature before measuring the luminescence. Then, 25 μl of Nano-Glo Live Cell Reagent (furimazine) was added to each well, and basal luminescence was measured using a luminometer (BioTek Inc., Winooski, VT, USA) for the first 10 min. Finally, cells were stimulated by adding 10 μl of SDF-1α at a final concentration of 100 ng/ ml to each well, and the real-time change of luminescence was measured for 30 min.