Problem	Possible cause	Solution
Roots look intact after enzyme incubation.	Enzyme medium has not been sufficiently mixed	Mix gently with a 1ml pipette several times. It can sometimes take up to 5–8 minutes to disaggregate root tips
Protoplast solution has many cell debris.	Harsh pipetting technique, or insufficient filtering	Pipette the protoplast-containing solution gently Filter the protoplast containing solution twice using a 40 μm cell strainer
Low protoplast yield	Not enough root tips were used or solution osmolarity is incorrect	Cut more primary root tips and add them to the media (at least 10) Check solution osmolarity
Protoplast pellet is lost during washing step	Centrifugation time is too short, or supernatant aspiration was not done immediately	Centrifuge at 500 g for 5 extra minutes Aspirate supernatant and perform washing step immediately after centrifugation
FACS flow stream get clogged	Protoplast concentration is too high	Dilute your protoplast sample at least 5 five times and sort again
Too many sorting events are detected.	Sample has many cell debris that are being detected as a sorting event	Determine appropriate gates in the forward vs side scatter plot. A live cell staining dye can be used to differentiate living cells from cell debris.