Fig. 1

A protocol to produce embryonic avian tracheal organoid cultures. Briefly, tracheas from 18-day-old chicken or 21- to 23-day old duck embryos were dissected, cut longitudinally, pooled, and treated with dispase to dissociate the epithelial cell lining. Epithelial cells were further dissociated by repeated pipetting. The sample was sieved, and epithelial cell sheets were separated from single cell contaminants by repeated centrifugation at low speed in phosphate buffered saline (PBS). The purified epithelial cells in basal medium were pelleted and resuspended in basement membrane extract (BME). Domes of 30 µL were assembled on 48-well plates and 200 µL airway organoid medium was added after solidification. Yellow circle icons indicate centrifugation steps. Created with BioRender.com