3 Methods
3.1 Preparation of
Protoplasts (See Notes
2 and 3)
The following methods are optimized for leaves and roots of Arabidopsis thaliana, and are adapted from references [14], [15], and
[17]. Seeds are sterilized and grown on vertical MS
+ sucrose plates
as previously described [49].
1. Harvest roots or aerial parts of the plants by using a
No. 22 scalpel, and then scraping them off the surface of the
agar. Collect tissues on a Kimwipe moistened with diH 2 O, and
weigh them. Transfer to a 60 Â 15 cm diameter plastic petri
dish, and add enzyme solution (solution BL for leaves and
solution BR for roots) in proportions of 200 mg tissue/5 mL
enzyme digestion medium.
2. Chop the tissues in the enzyme solution using a single-edged
razor blade, to produce tissue segments that are approximately
1 Â 1 mm (leaves) and 0.5 Â 0.5 mm (roots). Transfer to an
orbital shaker operating at 100 rpm; continue incubation for
60 min at room temperature.
3. After incubation, gently pipette the tissue fragments up and
down ten times, using a disposable plastic transfer pipette with
the end of the tip cut to produce a wider entry aperture
(approx. 4 mm). Filter the protoplast suspension through a
100 μm mesh cell strainer into a sterile polypropylene centrifuge tube (either 15 or 50 mL, depending on the volumes
employed in step 1).
4. Pellet protoplasts by centrifugation at 100 g for 3 min. All
further manipulations are done on ice.
5. Remove supernatant using a pipette, and gently resuspend the
protoplasts in 20.5% (w/v) sucrose dissolved in Solution A, to a
concentration of 3–10 Â 10
5 protoplasts/mL. Transfer to a
10 mL or 50 mL plastic centrifuge tube as appropriate for the
numbers of protoplasts. The lower (20.5% sucrose) phase
should not occupy more than 50% of the volume of the tube.
Gently overlay this phase with Solution A, to fill the tube to
about 90% of its total volume.
6. Centrifuge for 10 min at 500 g in a benchtop centrifuge
equipped with a swinging bucket rotor.
7. Collect protoplasts from the gradient interface using a transfer
pipette, and dilute with Solution A prior to sorting. If necessary, the protoplasts can be concentrated by centrifugation at
100 g for 3 min. Measure protoplast diameters by light microscopy using a hemocytometer.
Flow Cytometry and Sorting in Arabidopsis
273
3.1 Preparation of
Protoplasts (See Notes
2 and 3)
The following methods are optimized for leaves and roots of Arabidopsis thaliana, and are adapted from references [14], [15], and
[17]. Seeds are sterilized and grown on vertical MS
+ sucrose plates
as previously described [49].
1. Harvest roots or aerial parts of the plants by using a
No. 22 scalpel, and then scraping them off the surface of the
agar. Collect tissues on a Kimwipe moistened with diH 2 O, and
weigh them. Transfer to a 60 Â 15 cm diameter plastic petri
dish, and add enzyme solution (solution BL for leaves and
solution BR for roots) in proportions of 200 mg tissue/5 mL
enzyme digestion medium.
2. Chop the tissues in the enzyme solution using a single-edged
razor blade, to produce tissue segments that are approximately
1 Â 1 mm (leaves) and 0.5 Â 0.5 mm (roots). Transfer to an
orbital shaker operating at 100 rpm; continue incubation for
60 min at room temperature.
3. After incubation, gently pipette the tissue fragments up and
down ten times, using a disposable plastic transfer pipette with
the end of the tip cut to produce a wider entry aperture
(approx. 4 mm). Filter the protoplast suspension through a
100 μm mesh cell strainer into a sterile polypropylene centrifuge tube (either 15 or 50 mL, depending on the volumes
employed in step 1).
4. Pellet protoplasts by centrifugation at 100 g for 3 min. All
further manipulations are done on ice.
5. Remove supernatant using a pipette, and gently resuspend the
protoplasts in 20.5% (w/v) sucrose dissolved in Solution A, to a
concentration of 3–10 Â 10
5 protoplasts/mL. Transfer to a
10 mL or 50 mL plastic centrifuge tube as appropriate for the
numbers of protoplasts. The lower (20.5% sucrose) phase
should not occupy more than 50% of the volume of the tube.
Gently overlay this phase with Solution A, to fill the tube to
about 90% of its total volume.
6. Centrifuge for 10 min at 500 g in a benchtop centrifuge
equipped with a swinging bucket rotor.
7. Collect protoplasts from the gradient interface using a transfer
pipette, and dilute with Solution A prior to sorting. If necessary, the protoplasts can be concentrated by centrifugation at
100 g for 3 min. Measure protoplast diameters by light microscopy using a hemocytometer.
Flow Cytometry and Sorting in Arabidopsis
273