2.2 Protoplast
Isolation
1. Protoplast culture medium: 137 g/L sucrose, 4.3 g/L MS
(Murashige and Skoog Basal Salt Mixture), 0.75 g/L CaCl 2
·2H 2 O, 0.25 g/L NH 4 NO 3 , and 0.5 g/L MES hydrate,
pH 5.7 (with HCl). Sterilize by 0.22 μm filter.
2. 50 mL Falcon tubes (30/115 MM, sterile, conical bottom).
3. Enzyme solution: protoplast culture medium containing 1%
“ONOZUKA” RS (YAKULT HONSHA L0011), 0.2% Driselase (Sigma-Aldrich, St. Louis, MO), and 0.05% pectinase
(Worthington Biochemical Corporation, Lakewood, NJ),
pH 5.7 (with HCl). Prepare fresh when needed. Sterilize by
0.22 μm filter.
4. Laminar flow hood.
5. 0.22 μm Filters.
6. Eppendorf 022628168 5810R Refrigerated Centrifuge with
4 Â 750 mL Swing-Bucket Rotor, 15/50 mL Adapters.
7. Light microscope.
2.3 Protoplast
Transfection
1. Electroporation buffer: 2.4 g/L HEPES, 0.6 g/L
CaCl 2 ·2H 2 O, 6 g/L KCl, and 137 g/L sucrose. pH 7.2
(with KOH). Sterilize by 0.22 μm filter.
2. Peristaltic pump.
3. Maxi-prep plasmids encoding genes of interest tagged with
fluorescent proteins.
4. Bio-Rad Gene Pulser Xcell Electroporation System (Bio-Rad
Laboratories, Hercules, CA, cat. no. 165-2661).
5. 4 mm Electroporation cuvettes (Bio-Rad Laboratories,
Hercules, CA).
6. Leica SP8 laser scanning confocal microscopy (Leica TCS SP8)
or equivalent confocal including Zeiss LSM 880 with Airyscan
and Nikon A1 HD25/A1R HD25.
7. Leica 63x HC PL APO 63x/1.20 W (water immersion) CORR
CS2 objective lens.
8. Lasers (405 nm UV laser, 488 nm Blue laser, 552 nm Green
laser).
9. Leica HyD hybrid detector.
2.4 Super-Resolution
Confocal Microscopy
Imaging
1. ProLong™ Diamond Anti-Fade Mounting solution.
2. Nikon Structured Illumination Microscopy or equivalent
microscopy from Leica or Zeiss.
3. Nikon LU5 laser bed (488 and 561 nm laser lines).
4. Andor DU-897X-5254 EMCCD camera.
5. CFI SR Plan Apochromat IR 60 x WI (water immersion)
objective lens.
6. Nikon N-SIM system equipped with a Ti-E stand with Perfect
Focus.
Protoplasts Isolation and Transient Expression
159
Isolation
1. Protoplast culture medium: 137 g/L sucrose, 4.3 g/L MS
(Murashige and Skoog Basal Salt Mixture), 0.75 g/L CaCl 2
·2H 2 O, 0.25 g/L NH 4 NO 3 , and 0.5 g/L MES hydrate,
pH 5.7 (with HCl). Sterilize by 0.22 μm filter.
2. 50 mL Falcon tubes (30/115 MM, sterile, conical bottom).
3. Enzyme solution: protoplast culture medium containing 1%
“ONOZUKA” RS (YAKULT HONSHA L0011), 0.2% Driselase (Sigma-Aldrich, St. Louis, MO), and 0.05% pectinase
(Worthington Biochemical Corporation, Lakewood, NJ),
pH 5.7 (with HCl). Prepare fresh when needed. Sterilize by
0.22 μm filter.
4. Laminar flow hood.
5. 0.22 μm Filters.
6. Eppendorf 022628168 5810R Refrigerated Centrifuge with
4 Â 750 mL Swing-Bucket Rotor, 15/50 mL Adapters.
7. Light microscope.
2.3 Protoplast
Transfection
1. Electroporation buffer: 2.4 g/L HEPES, 0.6 g/L
CaCl 2 ·2H 2 O, 6 g/L KCl, and 137 g/L sucrose. pH 7.2
(with KOH). Sterilize by 0.22 μm filter.
2. Peristaltic pump.
3. Maxi-prep plasmids encoding genes of interest tagged with
fluorescent proteins.
4. Bio-Rad Gene Pulser Xcell Electroporation System (Bio-Rad
Laboratories, Hercules, CA, cat. no. 165-2661).
5. 4 mm Electroporation cuvettes (Bio-Rad Laboratories,
Hercules, CA).
6. Leica SP8 laser scanning confocal microscopy (Leica TCS SP8)
or equivalent confocal including Zeiss LSM 880 with Airyscan
and Nikon A1 HD25/A1R HD25.
7. Leica 63x HC PL APO 63x/1.20 W (water immersion) CORR
CS2 objective lens.
8. Lasers (405 nm UV laser, 488 nm Blue laser, 552 nm Green
laser).
9. Leica HyD hybrid detector.
2.4 Super-Resolution
Confocal Microscopy
Imaging
1. ProLong™ Diamond Anti-Fade Mounting solution.
2. Nikon Structured Illumination Microscopy or equivalent
microscopy from Leica or Zeiss.
3. Nikon LU5 laser bed (488 and 561 nm laser lines).
4. Andor DU-897X-5254 EMCCD camera.
5. CFI SR Plan Apochromat IR 60 x WI (water immersion)
objective lens.
6. Nikon N-SIM system equipped with a Ti-E stand with Perfect
Focus.
Protoplasts Isolation and Transient Expression
159