Journal: Theranostics

Article Title: Mechanically induced integrin ligation mediates intracellular calcium signaling with single pulsating cavitation bubbles

doi: 10.7150/thno.56813

Figure Lengend Snippet: Experimental design. (A) A schematic of experimental setup. A laser-induced single cavitation bubble (SCB) stimulates nearby cells in a glass-bottom dish, which is coated with 3 nm Ti and 7 nm Au to enhance laser absorption while allowing optical transmission for microscopy. (B) Top view of experimental setup. S d , standoff distance; γ, normalized standoff distance; R max , maximum bubble radius. (C) Superimposed bright-field and fluorescence image of three cells with different S d to a SCB with R max indicated by black dashed line. A mixture of stable Piezo1-knockout cells (P1KO) and cells transiently transfected with GFP-Piezo1 (P1TF) was used. (D) High-speed images of bubble dynamics (upper panel) and velocity field from particle image velocimetry (PIV) (lower panel). (E) Time evolution of the SCB-generated impulsive radial flow velocity along a horizontal axis (y = 0) through the bubble center. Outward velocity is positive, inward velocity is negative. The velocity amplitude decreases with γ. (F) Recording sequence: bright-field imaging of cell morphology, GFP imaging of Piezo1 expression, fluorescence imaging to simultaneously monitor intracellular Ca 2+ transients (340- and 380-nm excitation) and membrane poration (539-nm excitation), and high-speed imaging of SCB dynamics.

Article Snippet: At 48 h before SCB treatment and recording, P1KO cells were transiently transfected in a 6-well plate (the cells were seeded 8 h before transfection) in the presence of 10 μM ruthenium red and mouse Piezo1 (3 μg) using Fugene6 (Promega, Madison, WI).

Techniques: Transmission Assay, Microscopy, Fluorescence, Knock-Out, Transfection, Generated, Sequencing, Imaging, Expressing, Membrane

Journal: Theranostics

Article Title: Mechanically induced integrin ligation mediates intracellular calcium signaling with single pulsating cavitation bubbles

doi: 10.7150/thno.56813

Figure Lengend Snippet: Intracellular Ca 2+ response and PI uptake in SCB-stimulated cells. (A-D) Time series showing intracellular Ca 2+ response (top rows) and PI uptake (bottom rows) in representative individual cells with membrane poration (A, C) or without membrane poration (B, D), for Piezo1 knockout cells (P1KO) (A-B) and cells transiently expressing Piezo1 (P1TF) (C-D). Right: Single-shot bright-field images of cells before and after SCB treatment. The white arrow indicates the direction of radial expansion of the SCB at the location closest to the cell. Left: The cell contour before treatment is outlined with white dashed lines in the first PI image. Cells were exposed to SCB at t = 0. Scale bars, 30 µm. (E) Temporal profiles of averaged intracellular Ca 2+ response and PI uptake inside each cell shown in A-D. (F) and (G) Peak intracellular Ca 2+ response (circles) versus the normalized standoff distance for P1KO and P1TF cells. PI uptake values are color-coded. Almost all elicited Ca 2+ responses occurred at γ = 1-1.5. (H) Overall Ca 2+ response probability at γ = 1-1.5, including cells with an injury response (red), non-injury response (green), and no response/no injury (black) at different ranges of γ (1-1.2, 1.2-1.4, and 1.4-1.5). N indicates the number of cells in each group.

Article Snippet: At 48 h before SCB treatment and recording, P1KO cells were transiently transfected in a 6-well plate (the cells were seeded 8 h before transfection) in the presence of 10 μM ruthenium red and mouse Piezo1 (3 μg) using Fugene6 (Promega, Madison, WI).

Techniques: Membrane, Knock-Out, Expressing