7. Add 35 mL of electroporation buffer, slowly invert several
times for mixing, and then centrifuge at 80 Â g for 10 min
with no deceleration.
8. Repeat steps 7 and 8 twice or more, the bottom layer solution
should get clear and no obvious pellets should be viewed. If
not, repeat once more.
3.2 Protoplast
Transfection
1. Resuspend the protoplasts with appropriate volume of electroporation buffer and count cell number using hemocytometer
under light microscopy. Use electroporation buffer to dilute
the protoplast cells to obtain 2–5 Â 10
6 cell/mL cell density.
Generally, the volume of the protoplasts should be between
one-fifth and one-half of the total volume for a good transfection efficiency.
2. Add 500 μL resuspended protoplasts into 0.4 cm gap electroporation cuvettes (see Note 4).
3. Prepare 40 μg of DNA for each sample and dilute the DNA to a
final volume of 100 μL using electroporation buffer (see Note
5).
4. Mix the DNA and protoplasts gently and incubate them for at
least 10 min.
5. Electroporate the protoplasts with DNA at 130 V (voltage),
1000 μF (capacitance) for one pulse. The pulse time should be
in a range of 25–50 ms.
6. Incubate the electroporated protoplasts at room temperature
for 15 min.
7. Add 1 mL of protoplast culture medium to cuvette and transfer
the protoplast suspension to a petri dish. Add 1 mL of protoplast culture medium to the same petri dish.
8. Incubate the protoplasts in dark at 26
C for overnight
(12–24 h) period before observation (see Note 6).
3.3 Confocal
Imaging
1. Transfer the protoplasts suspension from petri dish to a 2 mL
Eppendorf tube.
2. The protoplasts will float up to the top after 10 min. Transfer
20 μL protoplasts from the top layer to a slide and cover it
carefully with coverslip. Then use confocal microscopy to analyze immediately (see Note 7).
3. Examine the transfected protoplasts using the Leica SP8 confocal microscope equipped with 63x HC PL APO 63x/1.20 W
(water immersion) CORR CS2 objective lens, Lasers (405 nm
UV laser, 488 nm Blue laser, 552 nm Green laser), and Leica
HyD hybrid detectors. As shown in Fig. 2a, b, upon their
co-expression in protoplasts, C-terminal GFP fusion protein
AtSar1c-GFP labeling the ERESs partially colocalizes (yellow
Protoplasts Isolation and Transient Expression
161
times for mixing, and then centrifuge at 80 Â g for 10 min
with no deceleration.
8. Repeat steps 7 and 8 twice or more, the bottom layer solution
should get clear and no obvious pellets should be viewed. If
not, repeat once more.
3.2 Protoplast
Transfection
1. Resuspend the protoplasts with appropriate volume of electroporation buffer and count cell number using hemocytometer
under light microscopy. Use electroporation buffer to dilute
the protoplast cells to obtain 2–5 Â 10
6 cell/mL cell density.
Generally, the volume of the protoplasts should be between
one-fifth and one-half of the total volume for a good transfection efficiency.
2. Add 500 μL resuspended protoplasts into 0.4 cm gap electroporation cuvettes (see Note 4).
3. Prepare 40 μg of DNA for each sample and dilute the DNA to a
final volume of 100 μL using electroporation buffer (see Note
5).
4. Mix the DNA and protoplasts gently and incubate them for at
least 10 min.
5. Electroporate the protoplasts with DNA at 130 V (voltage),
1000 μF (capacitance) for one pulse. The pulse time should be
in a range of 25–50 ms.
6. Incubate the electroporated protoplasts at room temperature
for 15 min.
7. Add 1 mL of protoplast culture medium to cuvette and transfer
the protoplast suspension to a petri dish. Add 1 mL of protoplast culture medium to the same petri dish.
8. Incubate the protoplasts in dark at 26
C for overnight
(12–24 h) period before observation (see Note 6).
3.3 Confocal
Imaging
1. Transfer the protoplasts suspension from petri dish to a 2 mL
Eppendorf tube.
2. The protoplasts will float up to the top after 10 min. Transfer
20 μL protoplasts from the top layer to a slide and cover it
carefully with coverslip. Then use confocal microscopy to analyze immediately (see Note 7).
3. Examine the transfected protoplasts using the Leica SP8 confocal microscope equipped with 63x HC PL APO 63x/1.20 W
(water immersion) CORR CS2 objective lens, Lasers (405 nm
UV laser, 488 nm Blue laser, 552 nm Green laser), and Leica
HyD hybrid detectors. As shown in Fig. 2a, b, upon their
co-expression in protoplasts, C-terminal GFP fusion protein
AtSar1c-GFP labeling the ERESs partially colocalizes (yellow
Protoplasts Isolation and Transient Expression
161