Fig. 2

Isolation of tomato chloroplasts on Percoll gradients. a Minimal disturbance of the 40–80% interface is critical for high yields of intact chloroplasts. To assemble the Percoll gradient, a pipette containing 40% Percoll is angled relative to the tube and placed immediately above the 80% Percoll cushion. The seal of the pipette and pipettor is slowly broken allowing gentle layering of the 40% Percoll solution over the 80% Percoll cushion. The pipette is slowly moved up the tube allowing layering with minimal disturbance of the 40–80% Percoll interface. The 40% Percoll solution is indicated as grey circles. b The location of cellular debris, broken chloroplasts, intact chloroplasts, and starch granules in 40–80% Percoll-Ficoll-PEG step gradient after centrifugation is shown. c The integrity of freshly isolated chloroplasts in 1X HS buffer was assessed using the Leica SP5 confocal microscope using standard FITC filters at 40× magnification. Scale bar = 10 µm