Monitoring Bitter Taste-Induced Calcium Signaling

A typical bitter taste signal can lead to an increase in intracellular calcium (Ca²⁺). This Ca²⁺ reaction is usually elicited via gustducin/PLCβ2 cascades, activation of voltage gated channel and transmitter release (13 (link), 17 (link)). Therefore, we investigated the activation of taste signaling by monitoring calcium mobilization. MGFs (1 per well × 10⁴ cells) were cultured overnight in 96-well black plates (Corning) with transparent bottoms. Cells were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS; Hyclone) containing calcium and magnesium, and then loaded with Fluo-4 AM (Thermo Fisher Scientific Inc., F14201) and F-127 (Thermo Fisher Scientific Inc., P3000MP) in the dark at room temperature for 1 hour. After washing with DPBS three times, the cells were incubated in darkness for another 30 minutes to completely de-esterify the dye. For single-cell calcium imaging, cells were examined using standard GFP filters (Olympus, IX83), and images were captured every 1 second for 140 seconds (inject salicin into the well at~10 seconds). 10 µM ATP (Solarbio) and DPBS were used as positive and negative controls, respectively. The baseline normalization curve of fluorescence intensity (F/F0; F0 was determined as the average of the first 5 readings) was drawn to time using GraphPad 8.0 (GraphPad Software Inc.).