Paraformaldehyde phosphate buffer solution

Sorted CD71⁺Ter119⁺ fetal liver erythroblasts from E11.5 embryos were attached to a slide glass using Shandon Cytospin 4 (Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque) for 15 min, permeabilized with 0.5% Triton X‐100 and 0.1% gelatin in PBS for 10 min and blocked with 0.1% gelatin and 2% goat‐serum (#143-06561; FUJIFILM Wako Pure Chemical) in PBS for 30 min at room temperature. Then cells were incubated with primary antibody against γ-H2AX (Ser139, #2577, Cell Signaling Technology) or GATA-1 (#3535, Cell Signaling Technology), followed by the incubation with secondary antibody conjugated with Alexa Fluor 568 (Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific). After washing, samples were mounted using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) and air-dried. Images were acquired using TCS SPE (Leica) and analyzed using ImageJ (NIH). The presence of micronuclei and γ-H2AX was determined manually by two independent investigators.

Plaque assay was performed as previously described^{29 (link)}. Briefly, 1 day before infection, Vero cells or VeroE6/TMPRSS2 cells (100,000 cells per well) were seeded into a 24-well plate and infected with SARS-CoV-2 (0.5, 5, 50, or 500 TCID₅₀) at 37 °C for 2 hours. A mounting solution containing 3% FBS and 1.5% carboxymethyl cellulose (Sigma-Aldrich, Cat#C4888-500G) was overlaid, followed by incubation at 37 °C. At 3 d.p.i., the culture medium was removed, and the cells were washed with PBS three times and fixed with 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque, Cat# 09154-85). The fixed cells were washed with tap water, dried, and stained with a staining solution [2% Crystal Violet (Nacalai Tesque, Cat# 09804-52) in water] for 30 minutes. The stained cells were washed with tap water and dried, and the size of the plaques was measured using Adobe Photoshop 2024 v25.0.0 (Adobe).

The morphologies of cell nuclei with and without vibrational stimulation were observed from fluorescent images of the cells. After removing the medium, cells were washed three times with 1 mL PBS. Next, cells were fixed with 4% paraformaldehyde (4% – Paraformaldehyde Phosphate Buffer Solution, Nacalai Tesque, Inc., Kyoto, Japan) in PBS for 10 min, washed with PBS, permeabilized with 0.1% Triton X‐100 (Triton X‐100 laboratory grade, Sigma‐Aldrich, MO, USA) in PBS for 5 min, and finally washed with PBS. After removing the PBS, the cells were stained with 0.05% Hoechst 33342 (H342, Dojindo Laboratories, Kumamoto, Japan) for 30 min and again washed with PBS. Then, fluorescence images of the cells were captured using the inverted microscope.
The captured fluorescent images were analyzed by fitting an ellipse to each cell nucleus using Image J. 300 cell nuclei near the gap edge were randomly selected to be analyzed. The angle of each cell nucleus was measured as the angle between the major axis of the fitted ellipse and a straight line parallel to the gap. The aspect ratio of each nucleus was measured as the ratio of the major axis to the minor axis of the fitted ellipse.