Fig. 1: Ultrasound as a remote trigger of calcium influx in HEK293 cells.

A A diagram illustrating the experimental setup: The ultrasound wave generator (MODUSON-2), connected to a power amplifier, is linked to an unfocused transducer (Olympus V318-SU), which was used for the stimulation of cultured cells. B The real-time imaging setup for monitoring Ca²⁺ influx during insonation. The ultrasound transducer was placed top-down in a well, submerged in a medium. The transducer’s position was secured with a 3D printed holder that fits into a 6-well plate or a 3.5- cm petri dish. C Ultrasound stimulation conditions with adjustable parameters controlled by wave generator: frequency, duration of the stimulation burst - stimulation time (ST) and resting time, pulse repetition frequency (PRF), duty cycle (DC), and amplitude are depicted. D–G The response of HEK293 cells to insonation. Various conditions of ultrasound pulse were tested on HEK293 cells stained with the Ca²⁺-sensitive dye. D Different ST (2.5, 5, and 10 s) with fixed 1 kHz PRF and 32% DC (n = 32 individual cells for each group), and (E) different DC (8, 16, and 32 %) with fixed ST (10 s) and PRF (1 kHz) were tested. n = 40 individual cells. F GdCl₃ (0.4 mM) inhibited the Ca²⁺ influx triggered by ultrasound (10 s ST, 1 kHz PRF, 32% DC). n = 36 individual cells for Gd³⁺ - and 40 for Gd³⁺+ group. G The Ca²⁺ oscillations are triggered by a consecutive repeated sequence of 10 s ST followed by a 110 s resting period. Changes in fluorescence intensity (FI) ratio (indicative of Ca²⁺ activity) during insonation are presented as a heatmap. Each row presents the Ca²⁺ activity of an individual cell (n = 34 individual cells). The FI ratio of CalRed at 488 nm-excitation over time was calculated for individual cells normalized to the pre-stimulated average FI ratio. The average FI ratios (with standard deviation) were calculated and are presented below the heatmap with black vertical lines indicating the periods of ultrasound stimulation. Source data are provided as a Source Data file. Items in (A) are adapted from ref. ³⁶ released under Creative Commons Attribution 4.0 International (CC BY 4.0).