Stabilizing Seahorse Assay Embryos

Tricaine (E10521, Merck, United States) was used to reduce embryo movement in the Seahorse well and to stabilize OCR, as previously shown by Raftery et al. (2017) (link). The embryos were incubated in a petri dish containing 125mg/ml Tricaine in E3 medium and then transferred into the Seahorse XF96 cell culture plates (101,085, Agilent, United States) with a 100μl cut pipet tip before adding 170μl of XF base medium with minimal DMEM (103,334, Agilent, United States) to each well. The central position of the embryos was checked with a microscope and corrected with a shortened Microloader™ pipet tip (EP5242956003, Eppendorf, Germany), if necessary. For analyzing the effect of sedation on the variability of OCR, Tricaine was injected via port A of the XF96 cartridge.