Protoplast Isolation and Imaging

Following the protocol of Wu et al. (2009) (link), 2 g of leaf tissue was incubated in 10 mL of enzyme solution (1% [w/v] cellulase from Trichoderma viride [Sigma], 0.25% [w/v] pectinase from Rhizopus spp. [Sigma], 0.4 M mannitol, 10 mM CaCl₂, 20 mM KCl, 0.1% [w/v] bovine serum albumin, and 20 mM MES at pH 5.7) for 1 h in light after placing Time Tape on the upper epidermis of the leaves and removing the lower epidermis of the leaves via Magic Tape. Protoplasts were then pelleted by centrifugation at 73 RCF for 3 min at 4°C using a Beckman J2-HS centrifuge and JS-13.1 rotor. Protoplasts were resuspended in a solution containing 0.4 M mannitol, 15 mM MgCl₂, and 4 mM MES at pH 5.7. eGFP fluorescence was visualized using protoplasts and leaves from stable eGFP plants with a Leica SP2 confocal microscope. The white light laser was used with 5% laser power and smart gain was adjusted to 100%. A 40X oil-emersion lens was used to visualize protoplasts and a ×20 objective lens was used to visualize intact cells from leaf samples. eGFP and chlorophyll were excited using a krypton/argon laser tuned to 488 nm, and eGFP and chlorophyll fluorescence were observed between the wavelengths of 500–520 nm and 660–700 nm, respectively.