This method can significantly speed up the Zebrafish embryo arraying procedure and make Zebrafish truly amenable for high throughput screening. The main advantage of this technique is the use of a 3D printed template to pre-array Zebrafish embryos into a 96 or 384 well matrix, which facilitates a highly efficient plate preparation process. We first had the idea for the method while performing the screening of nanoparticles by manually picking and selecting individual embryos into each well of a modular plate and wanted to increase efficiency.

In addition to Tianyu, fellow graduate student Yue Jiang will be demonstrating the procedure. Begin this procedure with design of the arraying template with a twelve by eight 96 well layout that fits a standard 96 well plate. As detailed in the text protocol.

Use a 3D printer with a 0.1 millimeter precision to print the template. Some key components of the template are the entrapment chamber, the entrapment well, the vacuum chamber, and the air-in outlet. To perform the Zebrafish embryo spawning, first separate males and females by a clear plastic divider.

Then, place two pairs of male and female fish per mating box one day prior to spawning. On the day of spawning, take off the dividers in the morning to mix male and female fish. Remove the male and female fish and collect Zebrafish embryos using a fine mesh strainer.

Wash the embryos with 250 milliliters of egg water. Transfer the collected embryos to Petri dishes with Holtfreter's Solution. Remove the dead and unfertilized embryos using a stereo microscope.

Place the embryos in a 28.5 degrees Celsius incubator. At four hours post-fertilization, observe the embryos and remove any dead and unhealthy embryos. The embryos are now ready for the next step.

Wash the template two to three times with 500 milliliters of deionized water before putting it into the drying oven for five minutes. Then, tape the bottom chamber with a piece sealing film. Connect a vacuum pump through the air outlet at the bottom of the template to generate negative pressure in the chamber sealed by the sealing film.

Using a plastic transfer pipette, place approximately 150 embryos into the template. Shake the entire template horizontally until each well has one embryo entrapped. With this step, it is critical to check and make sure on every available Zebrafish embryo.

If the Holtfreter buffer was very low, one could add additional buffer by shaking the template. Discard extra Holtfreter's Solution and embryos that are not entrapped in the wells. Turn off and disconnect the vacuum pump.

Place a standard 96 well plate upside down against the template and rotate both at the same time. Tap the bottom of the template or connect the air outlet to a compressed gas dusting can to transfer all trapped embryos from the template to the 96 well plate. Finally, remove the sealing film and wash the template three times from top to bottom with 500 milliliters of deionized water for future use.

Shown here is the partial image of a 96 well plate arrayed by the template. Conversely, this image shows the partial image of a 96 well plate arrayed manually. The overall health status of the embryos after being plated by manual and arraying template methods is displayed here.

Based on the hatching, survival, and malformation rates of the embryos, no significant effects on the overall health of the embryos were observed in either case. Once mastered, this technique will prepare one 96 or 384 well plate just in two to three minutes if performed properly. After its development, this technique paved the way for researchers to take full advantage of the new features of this model organism and truly achieve in vitro high throughput screening.