isolate
For isolating corneal epithelium, the corneas were subsequently digested at 37C for 90 mins with 2 mL of collagenase 2 and dispase II. Corneal stroma and corneal endothelium were then removed gently from the corneal epithelial sheet under stereo microscopy. The corneal epithelial sheet was further digested with 2mL TrypLE at 37C for 20 minutes with gentle pipetting to yield single cells. Epithelial cells were resuspended in 100L PBS containing 1g/mL PI solution. For isolating corneal stroma/endothelium, the corneas were cut into petal-shape and then digested at 37C for 60 mins with digest buffer. After incubation for digestion, corneas were mechanically dissociated using gentleMACS Dissociator and were filtered through a 70m nylon strainer. After the centrifugation, cells were resuspended in 100L PBS containing 1g/mL PI solution and filtered into a 5 mL round bottom tube. Single live cells of epithelium and stroma/endothelium were sorted on FACSAriaIII.
age
8 weeks old
sex
female
tissue
Cornea
BioSampleModel
Model organism or animal