Protoplast transfection

96-well plate protoplasts transfection

We developed a 96-well plate assay method, with Arabidopsis protoplasts, by adapting the method Sheen and colleagues reported (He, P. et al, J, 2007). We found that vortexing a 96-well plate after adding a polyethylene glycol (PEG) solution and centrifuging the plate after each washing step (see materials and methods for the details) allowed higher-efficient transformation with less number of protoplasts, compared to the original method, in our hands

Instrument


8-channel digital pipette	Centrifuge machine	U96 MicroWell Plates ( Nalge Nunc International)

digital vortex	Thermomixer R (Eppendorf AG,)	Washer ELx405^TM Microplate Washer (BioTek)

Material

polyethlyen glycol (PEG) solution: 40%(w/v) PEG4000 (Fluka, Buchs SG, Switzerland)
0.2 M mannitol
0.1 M CaCl₂

Protocol

10 μl of plasmid DNA solution (1 μg/μl) are dispensed into each well.
30 μl of protoplast suspension (1.5-2.5 x10⁵ cells/ ml) are dispensed into each well using a degital 8-channel pipette.
40 μl of a PEG solution are added to the wells.
Vortex with a digital vortex mixer at 800 rpm for 15 sec.
Incubate the plate for 10 min at room temperature.
200 μl of the W5 buffer are added in each well to reduce a viscosity of the solution in the wells.
The plate is then shaken in orbit at 900 rpm for 5 sec with Thermomixer R to mix the solution in the wells.
Centrifuge the plate at 100xg for 3 min at room temperature.
180 μl of the supernatant are removed from each well.
Wash two times in 150 μl of W5 buffer by consecutive resuspension and centrifugations.
Shake the plate with the Thermomixer R at 900 rpm for 5 sec
Incubate in the dark at 25 °C overnight (16 h).

Results: Transfect efficiency and gene expression

References: He, P., Shan, L., and Sheen, J. (2007). The use of protoplasts to study innate immune responses. Methods Mol Biol 354, 1-9.