Facsmax cell dissociation solution

Embryos at 20 hpf were briefly washed in calcium-free Ringer’s solution and deyolked by up and down pipetting. Deyolked embryos were pelleted by 500 ×g centrifugation for 5 min. They were briefly washed with FACSmax cell dissociation solution (Genlantis) and transferred in a 60 mm petri dish with fresh FACSmax solution, then incubated at 28.5°C. Single-cell dissociation was carefully monitored every 5 min and was generally achieved within 30 min of incubation. Efficient dissociation was helped by firmly tapping the petri dish and by gentle pipetting. Single cells were exhaustively washed twice in cold PBS, pelleted and resuspended in 500 μL of cold PBS. Single cells were filtered in a Falcon tube with a cell strainer cap (Fisher Scientific) and placed on ice until cell sorting. Sorting was performed using a BDARIA IIIu FACS with DIVA 8 sofware (BD Biosciences San Jose, CA, USA). GFP expressing cells were identified using a 488 nm laser and a 530/30 BP filter. Cells were collected in cold PBS on ice.

β-catCA-mosaically introduced embryos were prepared as described above. HS:GFP-2A-β-catCA-transgenic zebrafish embryos were used as β-catCA-ubiquitously introduced embryos. Cell dissociation was performed using established protocol^{59 (link)} with the following modification: 40 embryos per group at 8.3 hpf stage were placed in a solution of 2 mg/ml Pronase (Roche, Upper Bavaria, Germany) in E2 (15 mM NaCl, 0.5 mM KCl, 2.7 mM CaCl₂, 1 mM MgSO₄, 0.7 mM NaHCO₃, 0.15 mM KH₂PO₄, and 0.05 mM Na₂HPO₄) on a 2% agar-coated dish for ~6 min at 28.5 °C. After dechorionation, embryos were washed with deyolking buffer (1/2 Ginzburg Fish Ringer without calcium: 55 mM NaCl, 1.8 mM KCl, and 1.25 mM NaHCO₃). Embryos were transferred into a 1.5 ml tube and then the yolk was disrupted by pipetting with a 1000 μl tip. The embryos were shaken for 5 min at 2 g to dissolve the yolk (Thermomixer, Eppendorf, Hamburg, Germany). Cells were pelleted at 300 g for 1 min and the supernatant discarded. Cell pellets were resuspended in FACSmax Cell Dissociation Solution (Genlantis, San Diego, CA). β-catCA-expressing (GFP⁺) cells were sorted by FACSAriaII (BD, Franklin Lakes, NJ). Uninjected negative control cells were also sorted under similar conditions. Sorted cells were pelleted and dissolved in TRIzol reagent (Invitrogen, Waltham, MA).

A thousand 123count ebeads (Biosciences #01-1234-42) were added to embryos at 24 hours post-fertilization (hpf). They were briefly washed in calcium-free Ringer’s solution and de-yolked by up and down pipetting. De-yolked embryos were pelleted by 500 × g centrifugation for 5 min. They were briefly washed with FACSmax cell dissociation solution (Genlantis) and transferred in a 60 mm petri dish with FACSmax solution, then incubated at 28.5 °C. Single-cell dissociation was carefully monitored every 5 min and was generally achieved within 30 min of incubation. Efficient dissociation was helped by firmly tapping the petri dish and by gentle pipetting. Single cells were exhaustively washed twice in cold PBS, pelleted and resuspended in 500 μL of cold PBS. Single cells were filtered in a Falcon tube with a cell strainer cap (Fisher Scientific) and placed on ice until counting. Samples were analyzed using a LSRII flow cytometer with DIVA 8 software (BD Biosciences San Jose, CA, USA). GFP expressing cells were identified using a 488 nm laser and a 530/30 BP filter and 123count ebeads with a 561 laser and a 610/20 filter and a 405 laser with a 525/50 filter. 123count ebeads are completely separated from cells on a FSC/SSC dot plot.