Journal: Communications Biology

Article Title: High frequency acoustic cell stimulation promotes exosome generation regulated by a calcium-dependent mechanism

doi: 10.1038/s42003-020-01277-6

Figure Lengend Snippet: a Perspective and side view schematics (see also images in Supplementary Fig. ) of the experimental set-up in which the SRBW (not to scale), generated along a piezoelectric lithium niobate (LiNbO ) substrate by applying an AC electric signal at the device’s resonant frequency (10 MHz) to an interdigitated transducer electrode (IDT) photolithographically patterned on the substrate, is coupled through a thin layer of silicon oil into a glass-bottom culture plate containing the adherent cells to stimulate their production of exosomes. b Acetylcholine esterase activity of EVs isolated from spent U87-MG cell media as a function of the post-excitation (i.e., after 10 min of SRBW exposure) incubation time relative to that for the control sample, which comprised unexposed cells incubated over the same period. c Enhancement in U87-MG EV production under SRBW excitation with increasing input power to the device, as indicated by the increase in esterase activity in the spent cell media relative to the unexposed control; the post-excitation incubation period in all cases was fixed at 30 min. Cell viability data at higher powers beyond 4 W are shown in Supplementary Fig. . d U87-MG cell viability, as measured from an MTT assay, e U87-MG cell population density and f relative esterase activity of U87-MG (red squares) and A549 (blue circles) EVs following successive 10-min excitation and 30-min incubation cycles n ( n = 1: no shading, n = 4: fine shading, n = 7: coarse shading). The data are represented in terms of the mean value ± the standard error over triplicate runs, and the asterisks *** and **** indicate statistically significant differences with p < 0.001 and p < 0.0001, respectively. The corresponding results for A549 cells can be found in Supplementary Figs. and , although we have included the A549 cell esterase activity data for successive cycles in f .

Article Snippet: Ltd., Gulfview Heights, SA, Australia), A549 and U87-MG cells (American Type Culture Collection, Manassas, VA, USA), Exosome Spin Columns (MW 3000), T25 cell culture flask, MatTek 24-well glass-bottom plates and Nunc TM Lab-Tek TM II Chamber Slide and Chambered Coverglass were acquired from Thermo Fischer Scientific Pty.

Techniques: Generated, Activity Assay, Isolation, Incubation, Control, MTT Assay

Journal: Communications Biology

Article Title: High frequency acoustic cell stimulation promotes exosome generation regulated by a calcium-dependent mechanism

doi: 10.1038/s42003-020-01277-6

Figure Lengend Snippet: Comparison of a the number concentration, obtained through NTA, b the hydrodynamic size distribution, obtained through DLS, c representative cryo-EM images, d the protein profile, obtained via western blotting, and e the band intensities of EVs isolated from the unexposed control (dark grey bars in b ) and SRBW-treated (light grey bars in b ) U87-MG cells (aside from a , e where blue circles indicate EVs isolated from A549 cells and red squares indicate EVs from U87-MG cells) after 7 successive excitation–incubation cycles; the scale bars in c represent a length of 50 nm. The data are represented in terms of the mean value ± the standard error over triplicate runs, and the asterisks *, ** and *** indicate statistically significant differences with p < 0.05, p < 0.01 and p < 0.001, respectively. We note that the blot image for syntenin-1 has been spliced, as demarcated with a dividing line; a full scan of the entire blot is provided in Supplementary Fig. .

Article Snippet: Ltd., Gulfview Heights, SA, Australia), A549 and U87-MG cells (American Type Culture Collection, Manassas, VA, USA), Exosome Spin Columns (MW 3000), T25 cell culture flask, MatTek 24-well glass-bottom plates and Nunc TM Lab-Tek TM II Chamber Slide and Chambered Coverglass were acquired from Thermo Fischer Scientific Pty.

Techniques: Comparison, Concentration Assay, Cryo-EM Sample Prep, Western Blot, Isolation, Control, Incubation

Journal: Communications Biology

Article Title: High frequency acoustic cell stimulation promotes exosome generation regulated by a calcium-dependent mechanism

doi: 10.1038/s42003-020-01277-6

Figure Lengend Snippet: Exosome protein profiling via a western blot analysis, showing the progressive increase in the relative band intensity of b ALIX, c syntenin-1 and d CD63 with successive number of excitation–incubation cycles n , compared to the unexposed control over the same duration. e , f RT-qPCR analysis quantifying the mRNA expression of ALIX and CD63 between the unexposed control and SRBW-irradiated A549 (blue circles) and U87-MG (red squares) cells after successive number of cycles, normalised against GAPDH. The data are represented in terms of the mean value ± the standard error over triplicate runs (10 runs for the RT-qPCR experiments), and the asterisks *, ** and **** indicate statistically significant differences with p < 0.05, p < 0.01 and p < 0.0001, respectively.

Article Snippet: Ltd., Gulfview Heights, SA, Australia), A549 and U87-MG cells (American Type Culture Collection, Manassas, VA, USA), Exosome Spin Columns (MW 3000), T25 cell culture flask, MatTek 24-well glass-bottom plates and Nunc TM Lab-Tek TM II Chamber Slide and Chambered Coverglass were acquired from Thermo Fischer Scientific Pty.

Techniques: Western Blot, Incubation, Control, Quantitative RT-PCR, Expressing, Irradiation

Journal: Communications Biology

Article Title: High frequency acoustic cell stimulation promotes exosome generation regulated by a calcium-dependent mechanism

doi: 10.1038/s42003-020-01277-6

Figure Lengend Snippet: a Intracellular Ca 2+ levels, measured from a Fura-2 AM assay, b acetylcholine esterase activity and, c mRNA expression of ALIX and CD63, as quantified via RT-qPCR analysis, for A549 (blue circles) and U87-MG (red squares) cells exposed to the SRBW irradiation compared to the unexposed control (i), in the absence (−) and presence (+) of extracellular calcium as well as a combination of a Ca 2+ inhibitor (thapsigargin), ion channel blocker (amiloride HCl) and membrane-permeable intracellular Ca 2+ chelator (BAPTA-AM); see the key in Table . The data are represented in terms of the mean value ± the standard error over quadruplicate runs, and the asterisks *, **, *** and **** indicate statistically significant differences with p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively.

Article Snippet: Ltd., Gulfview Heights, SA, Australia), A549 and U87-MG cells (American Type Culture Collection, Manassas, VA, USA), Exosome Spin Columns (MW 3000), T25 cell culture flask, MatTek 24-well glass-bottom plates and Nunc TM Lab-Tek TM II Chamber Slide and Chambered Coverglass were acquired from Thermo Fischer Scientific Pty.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Irradiation, Control, Membrane