Plant cell protoplast isolation
Grow plants of Arabidopsis thaliana ( ecotype Col0 ) on a 3 : 1 ( v / v ) compost to vermiculite mixture at 20oC , under short day conditions ( 12h light : 12h dark ) and low light intensities ( 50 - 100µE m - 2 s - 1 ) .
Prepare fresh enzyme solution and transfer to glass petridish .
Remove 20 - 40 leaves from 4 week old Arabidopsis thaliana rosettes .
Pile up 5 - 10 leaves at a time , cut into thin ( ~ 2mm ) strips across the leaf using a sharp scapel blade dipped into ethanol .
Place strips into enzyme solution .
Repeat until all leaves have been cut .
Vacuum infiltrate the enzyme solution .
Leave cells in the petri dish at 20oC without shaking .
Twice filter cells and enzyme solution through a 125μm nylon mesh to remove residual debris into a pointed bottom , glass test tube .
Leave cells to settle to the bottom of the tube .
Carefully aspirate out the liquid solution leaving the cell pellet intact .
Add Incubation Buffer .
Gently resuspend protoplast in incubation solution by light swirling of the test tube .
Check protoplast integrity and determine yield using a disposable hemocytomer .
