Liberase enzyme blend

Larvae were washed twice using artificial seawater. The larvae were washed in artificial seawater supplemented with 25 mM EGTA, then lysed for 1 h in Triton X-100 1% in the same buffer on ice. We could observe the appearance of a gel containing bristles. The gel was washed twice in 10 mM Tris-HCl, 2.5 mM MgCl₂, 0.5 mM, CaCl₂ pH 7.6. 50 µL were left in the sample at the last wash, and the gel was digested with 5 U of DNase I (Thermo Fischer Scientific) for 2.5 h at 37 °C with 360 rpm agitation. Next, the digest was supplemented with 100 µL of artificial seawater and digested with 200 µL of 2.5 µg/µL Liberase Enzyme Blend (Sigma-Aldrich) for 1 h at 50 °C. The bristles were washed three times in artificial seawater. The isolated bristles were mounted on µ Slide I Luer (Ibidi). The refractive index tomograms of isolated bristles were obtained by Nanolive 3D Cell Explorer, and raw data were deposited at 10.5281/zenodo.10207240.

A 12-mm-diameter circle from the skin at the site of cell injection was harvested 5 days after transplantation and then minced using a blade. To achieve single-cell isolation, tissue samples were digested with Liberase™ enzyme blend (Sigma-Aldrich, Oakville, ON, Canada) at 37°C for 45 minutes in Hank’s Balanced Salt Solution with 250 r/min shaking. Digestion was stopped by adding RPMI media supplemented with 10% fetal bovine serum (FBS) and the cell suspension passed through a 40-µm pore size cell strainer and washed with same media.