*2.7. Calcein Uptake*

To assess if the cell membrane had been permeabilised, a nontoxic cell-impermeable fluorescent dye, calcein, was added during treatment with US± MB. A 50 mg/mL calcein stock solution in 1 M NaOH was kept at 2–8 ◦C protected from light. Immediately prior to treatment with US ± MB, calcein was mixed with NaCl 9 mg/mL ± MBs in the 1 mL that was injected into the Petaka. A concentration of 6 µM calcein was used in all experiments.

After treatment with US ± MB, the cells were incubated for 1 h to allow for cell membrane pores to re-seal [4,33], flushed twice with phosphate-buffered saline (PBS), detached using trypsin-EDTA 0.05% and harvested from the Petaka. Following centrifugation and resuspension in PBS, the cells were analysed by flow cytometry on an Accuri C6 flow cytometer (BDBioscience, Franklin Lakes, NJ, USA). Data collected from Acurri C6 were gated in FlowJo®, and the uptake of calcein was measured as a percentage of calcein-positive cells. The gating strategy is shown in Supplementary Figure S1. The median fluorescence intensity (MFI) of the cells in the calcein-positive population was also recorded.
