Fig 1. Schematic diagram of the hydrostatic pressure device and the chilling curve applied for zebrafish embryos.
(A) The capacity of the pressure chamber is 100 cm3, the range of use is between 2 MPa and 90 MPa. The custom-made software runs the adjusted or cell-specific preconditioning treatment by controlling hydrostatic pressure build up, holding and coming back to atmospheric pressure and the temperature in the pressure chamber. The biological material is placed into an appropriate cell container (e.g. luer lock syringe) filled with the convenient fluid (this case by system water). After proper closing cell container is merged into the pressure filled with water. (B) Groups of 26-somites or Prim-5 stage embryos were placed on ice in zebrafish system water containing cryoprotectants. Cooling occurred according to the following cooling rates: 3.16°C/min for 4.5 minutes, 0.28°C/min for 22.5 minutes, 0.06°C/min for 30 minutes, and 0.01°C/min for 33 minutes. Embryos were kept at 0.0–0.3°C for additional 22.5 hours (24 hours in total).