In this paper we demonstrate that contactless high-frequency ultrasound microbeam stimulation (HFUMS) is capable of eliciting cytoplasmic calcium (Ca2+) elevation in human umbilical vein endothelial cells (HUVECs) and the associated mechanisms were highly correlated with those of shear force induced cytoplasmic Ca2+ elevation. Ca2+ and cytoplasmic Ca2+stores were involved in the Ca2+ elevation. These results suggested that HFUMS may be potentially a novel non-contact tool for studying cellular mechanotransduction if the acoustic pressures at such high frequency could be quantified. < 0.05. The number of cells was quantitated from over triplicate experiments. Results Cytoplasmic Ca2+ variations in HUVECs elicited by HFUMS Live-cell fluorescence imaging was performed to monitor Ca2+ changes in HUVECs stained with Fluo-4 AM due to HFUMS. It was observed that HFUMS elicited significant fluorescence increases in HUVECs (Fig. 2.a) (Supplementary video 1 and Supplementary video 2). Fig. 2.b illustrates the normalized Ca2+ temporal variations in HUVECs due to HFUMS. The HUVECs clearly exhibited transient Ca2+ elevations when HFUM was on and then the Ca2+ level in HUVECs was gradually reduced by ~ 1.25 at 240 s. In addition the calcium elevations were further quantitated using the program described previously for the quantitative analysis. The CRI value for HUVECs stimulated by HFUM was ~1.89 whereas the CRI value MK-0517 (Fosaprepitant) for control cells (without HFUMS) was 0 (n=21) (Fig. 2.c). Therefore these results clearly demonstrated that HFUMS elicited significant Ca2+ elevations in HUVECs compared to the control cells. Fig. 2 Cytoplasmic Ca2+ changes in HUVECs due to HFUMS Effect of HFUM exposure on cytoplasmic Ca2+ elevation in HUVECs Dependences of HFMUS-elicited cytoplasmic Ca2+ elevation on both input voltage to the transducer and duty cycle of the input bursts were examined. Note that acoustic pressure generated from the transducer is proportional to the input voltages to the transducer if the input power level is low (Johns et al. 2007). Fig. 3.a demonstrates the CRIs for HUVECs at the simulated acoustic power at the given input voltages. When the simulated acoustic pressure was 1.8 and 2.4 MPa the normalized CRI values slightly increased up to 0.18 and 0.36 from the base-line (acoustic pressure = 0 MPa) respectively. In contrast the CRI values at the simulated acoustic pressure of 3.0 and 3.6 MPa significantly increased up to almost three-fold over the CRI value at the simulated acoustic pressure Calcrl of 2.4 MPa (p-value = 0.028 < 0.05). In this experiment the CRI for HUVECs was highest when the simulated acoustic pressure was 3.6 MPa. In addition we examined the dependence of calcium response of the cells to HFUMS on the duty cycle of input bursts. We here examined the CRI values for HUVECs at the lower duty cycles than 1 % (simulated acoustic pressure of 3.6 MPa) since few cells were detached from the cell culture dish by HFUMS at the simulated acoustic pressure of MK-0517 (Fosaprepitant) 3.6 MPa and the duty cycle of 1%. Fig. 3 Normalized CRI values for HUVECs at the simulated acoustic pressure at given input voltages MK-0517 (Fosaprepitant) (0 9.48 12.64 15.8 and 18.96 Vpp) and duty cycles at the stimulated acoustic pressure of 3.6 MPa The CRI values for HUVECs increased as a function of duty cycles as shown in Fig. 3.b. When the duty cycle was 0.10 %10 % at 3.6 MPa HUVECs did not exhibit any notable Ca2+ elevation. In contrast the mean of CRI increased as the duty cycles further increased. The normalized CRI values at the duty cycles of 0.25 and 0.50 % were measured to be 0.41 and 0.78 respectively. MK-0517 (Fosaprepitant) Altogether these results demonstrated that there was indeed a dose-response relationship between the CRI values and acoustic pressure in HUVECs. Effects of extracellular calcium on HFUMS-induced Ca2+ elevations in HUVECs Previous studies showed that upon mechanical stimulation of HUVECs HUVECs exhibited cytoplasmic Ca2+ elevations. It is however important to note that the cytoplasmic Ca2+ elevations were affected by both extracellular calcium influx and Ca2+ release from cytoplasmic calcium stores (Yamamoto MK-0517 (Fosaprepitant) et al. 2000; Nishitani et al. 2011). To determine the origin of cellular Ca elevation we first examined whether the extracellular Ca2+ effects on HFUMS-induced Ca2+ elevation in HUVECs. Both the calcium chelation of EDTA and GdCl3 significantly MK-0517 (Fosaprepitant) inhibited calcium elevations in HUVECs (Fig. 4) due to HFUMS compared to those in the control cells (Fig. 2.a). The spontaneous calcium increases as shown in the untreated cells were not observed in the majority of the cells treated with EDTA and Gadolinium (III) chloride. The CRI values for HUVECs treated.