Isolation of GFP-Positive Cells from Zebrafish Embryos

Embryos from required stock were grown up to the desired stage (from 14 to 72 hpf) in standard embryo media at 29 °C. To prevent melanisation in embryo melanocytes, PTU (N-Phenylthiourea, Sigma-Aldrich, Cat. No. P7629) was added at a final concentration of 0.003% at 24 hpf. To stimulate eGFP expression, embryos were heat-shocked by placing them in 42 °C embryo media followed by 1 h incubation at 37 °C and at least 1 h incubation at 29 °C. If required, the embryos were dechorionated using pronase (Pronase from Streptomyces griseus, Sigma-Aldrich, Cat. No. 000000010165921001) at a final concentration of 1 mg/ml⁸⁰ . The heads were cut from all the embryos at stage 30 hpf or older to decrease the number of sox10-positive cells of craniofacial skeletal and otic fates. Embryos were then digested as previously described with small modifications^{81 (link)}. In brief, embryos were rinsed with Ca-, Mg- DPBS (Sigma-Aldrich, D8537), placed in a flask containing TrypLE™ Express Enzyme (ThermoFisher Scientific, Cat. No. 12605036) in ratio of 10 ml per 100 embryos, containing 0.003% Tricaine (Sigma-Aldrich, Cat. No. E10521); incubated for 30–90 min at 100 rpm, 37 °C in the shaker incubator with constant monitoring until the embryos were digested to a mixture of single cells and small fragments of tissue; then digestion mixture was triturated 10–15 times, using a Pasteur pipette; passed through 100-micron strainer (MACS SmartStrainers, Miltenyi Biotech., Cat. No. 130-098-463,) into 50 ml Falcon tube and centrifuged for 5 min, 500 x g, 4 °C. The cell pellet was re-suspended in DPBS and the cell suspension was passed through 30-micron strainer (MACS SmartStrainers, Miltenyi Biotech. Cat. No. 130-110-915) into 50 ml Falcon tube and centrifuged again for 5 min, 500 x g, 4 °C following by re-suspending the cells in 0.5–1 ml cell isolation media (2% FCS, DPBS:HBSS = 1:1 and 1 mM SYTOX Blue Dead Cell stain (ThermoFisher Scientific, S34857)). Cells were imaged before FACS and after FACS to confirm successful purification of GFP+cells.