Janus green

The expression of TRPV4 was measured using the In-cell ELISA Kit (Thermo), as described in the manufacturer’s protocol. The bEnd.3 cells were seeded in a 96-well culture plate at a density of 2.4 × 10⁴ cells/well. After 18 h of incubation, cells were washed with PBS, then the wells were filled with medium and sealed without bubbles by microplate sealing tape. Shockwave treatment was conducted as previously described. At 18 h post-shockwave treatment, the cells were washed twice with PBS and fixed with 4% formaldehyde for 15 min at room temperature. The cells were incubated with permeabilization buffer (0.1% triton X-100 in Tris-buffered saline (TBS)) for 15 min at room temperature and then the plate was washed twice with TBS. Peroxidase suppressor (Thermo) was then added to each well and incubated for 20 min. The washing steps were repeated and the plate was incubated with blocking buffer at room temperature for 30 min. After blocking, plates were incubated with TRPV4 antibody (Thermo) overnight at 4 °C followed by HRP conjugated secondary antibody for 1 h at room temperature. The plates were washed and incubated with TMB substrate. After sufficient blue color development, stop solution was added to each well and absorbance was measured at 450 nm. Results across wells were then normalized to cell number based on whole-cell staining with Janus Green (Thermo).