At low-intensity levels, ultrasound can potentially generate therapeutic effects on living cells, and it can trigger sonoporation when microbubbles (MBs) are present to facilitate drug delivery. Yet, our foundational knowledge of low-intensity pulsed ultrasound (LIPUS) and sonoporation remains to be critically weak because the pertinent cellular bioeffects have not been rigorously studied. In this article, we present a populationbased experimental protocol that can effectively foster investigations on the mechanistic bioeffects of LIPUS and sonoporation over a cell population. Walkthroughs of different methodological details are presented, including the fabrication of the ultrasound exposure platform and its calibration, as well as the design of a bioassay procedure that uses fluorescent tracers and flow cytometry to isolate sonicated cells with similar characteristics. An application example is also presented to illustrate how our protocol can be used to investigate the downstream cellular bioeffects of leukemia cells. We show that, with 1-MHz LIPUS exposure (with 29.1 J/cm 2 delivered acoustic energy density), variations in viability and morphology would be found among different types of sonicated leukemia cells (HL-60, Molt-4) in the absence and presence of MBs. Taken altogether, this article provides a reference on how cellular bioeffect experiments on LIPUS and sonoporation can be planned meticulously to acquire strong observations that are critical to establish the biological foundations for therapeutic applications.

Cell membranes are susceptible to biophysical damages. These biophysical damages often present themselves in challenging oxidative environments, such as in chronic inflammation. Here we report the damage evolution after single myoblasts were individually subjected to femtosecond (fs) laser photoporation on their plasma membranes under normal and oxidative conditions. A well-characterized tunable fs laser was coupled with a laser scanning confocal microscope. The post-damage wound evolution was documented by real-time imaging. The fs laser could generate a highly focused hole at a targeted site of the myoblast plasma membrane. The initial hole size depended on the laser dosage in terms of power and exposure duration. With the same laser power and irradiation duration, photoporation invoked bigger holes in the oxidative groups than in the control. Myoblasts showed difficulty in repairing holes with initial size beyond certain threshold. Within the threshold, holes could apparently be resealed within 100 s under the normal condition; while in oxidative condition, the resealing process could take 100-300 s. The hole-resealing capacity of myoblasts was compromised under oxidative stress particularly when the oxidative exposure was chronic. It is interesting to note that brief exposure to oxidative stress apparently could promote resealing in myoblasts after photoporation.

Sonoporation via microbubble-mediated ultrasound exposure has shown potential in drug and gene delivery. However, there is a general lack of mechanistic knowledge on sonoporation-induced cellular impact after membrane resealing, and this issue has made it challenging to apply sonoporation efficiently in practice. Here, we present new evidence on how sonoporation, without endangering immediate cell viability, may disrupt downstream cellular hemostasis in ways that are distinguished from the bioeffects observed in other sonicated and unsonoporated cells. Sonoporation was realized on HL-60 leukemia cells by delivering pulsed ultrasound (1 MHz frequency, 0.50 MPa peak negative pressure; 10% duty cycle; 30 s exposure period; 29.1 J/cm2 acoustic energy density) in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio). Results showed that 54.6% of sonoporated cells, despite remaining initially viable, underwent apoptosis or necrosis at 24 h after sonoporation. Anti-proliferation behavior was also observed in sonoporated cells as their subpopulation size was reduced by 43.8% over 24 h. Preceding these cytotoxic events, the percentages of sonoporated cells in different cell cycle phases were found to be altered by 12 h after exposure. As well, for sonoporated cells, their expressions of cytoprotective genes in the heat shock protein-70 (HSP-70) family were upregulated by at least 4.1 fold at 3 h after exposure. Taken altogether, these findings indicate that sonoporated cells attempted to restore homeostasis after membrane resealing, but many of them ultimately failed to recover. Such mechanistic knowledge should be taken into account to devise more efficient sonoporation-mediated therapeutic protocols.