the samples, estimation of proportions of different cell types and
other fluorescent objects, and general determination of cellular
integrity/viability. Microscope technologies have advanced, and
costs continue to decrease. The EVOS FLoid™ Cell Imaging
Station from ThermoFisher employs light-emitting diodes for fluorescence excitation (Blue channel: 390/40 nm; Green channel:
482/18 nm; Red channel: 586/15 nm), at a very competitive
price.
Specifically for agnostic sampling of expression states, and for
any other application that envisages parallel processing of populations of single cells or nuclei, specialized instruments are needed. In
this review, we also describe the use of the Chromium Controller
(10ÂGenomics; Pleasanton, California) for this purpose.
2 Materials
General Information and Precautions: Always wear a laboratory
coat, disposable gloves, and protective eyewear. Clean the working
areas with 70% ethanol, before and after use. All chemicals are
reagent grade, unless otherwise noted. The following section lists
specialty chemicals, kits, and equipment needed for the described
applications and methods.
2.1 Preparation of
Protoplasts
1. Polysaccharide hydrolases: Cellulysin, Pectolyase, Cellulase RS,
Macerozyme R10.
2. Solution A: For 200 mL solution, mix 2 mL 1 M KCl, 400 μL
1 M MgCl 2 , 400 μL 1 M CaCl 2 , 0.2 g BSA, 78 mg 2-(N-morpholino) ethanesulfonic acid hydrate (MES), and 21.86 g mannitol (giving the following concentrations: 10 mM KCl, 2 mM
MgCl 2 , 2 mM CaCl 2 , 1 mg/mL BSA, 2 mM MES, 0.6 M
mannitol). Adjust pH to 5.5 with 1 M Tris base; typically, less
than 50 μL is required.
3. Solution BL (for leaves; 50 mL): 50 mL Solution A, 750 mg
Cellulysin (1.5% w/v), and 50 mg Pectolyase (0.1% w/v).
Prepare immediately prior to tissue harvest.
4. Solution BR (for roots; 50 mL): 50 mL Solution A, 750 mg
Cellulase RS (1.5% w/v), and 100 mg Macerozyme R10 (0.2%
w/v). Prepare immediately prior to tissue harvest.
5. 20.5% (w/v) sucrose dissolved in Solution A.
6. Supplies: BD Falcon™ or Sysmex 40, 70, 100 μm pore size
disposable mesh filters; disposable plastic transfer pipettes;
No. 22 scalpel; single-edged razor blades honed to a thickness
of 0.2 mm (we recommend VWR no. 55411-050). Kimwipes
paper tissues or similar. Plastic petri dishes (60 Â 15 cm).
Hemocytometer. BD Falcon™ 15 and 50 mL plastic centrifuge
tubes.
270
David W. Galbraith and Guiling Sun
other fluorescent objects, and general determination of cellular
integrity/viability. Microscope technologies have advanced, and
costs continue to decrease. The EVOS FLoid™ Cell Imaging
Station from ThermoFisher employs light-emitting diodes for fluorescence excitation (Blue channel: 390/40 nm; Green channel:
482/18 nm; Red channel: 586/15 nm), at a very competitive
price.
Specifically for agnostic sampling of expression states, and for
any other application that envisages parallel processing of populations of single cells or nuclei, specialized instruments are needed. In
this review, we also describe the use of the Chromium Controller
(10ÂGenomics; Pleasanton, California) for this purpose.
2 Materials
General Information and Precautions: Always wear a laboratory
coat, disposable gloves, and protective eyewear. Clean the working
areas with 70% ethanol, before and after use. All chemicals are
reagent grade, unless otherwise noted. The following section lists
specialty chemicals, kits, and equipment needed for the described
applications and methods.
2.1 Preparation of
Protoplasts
1. Polysaccharide hydrolases: Cellulysin, Pectolyase, Cellulase RS,
Macerozyme R10.
2. Solution A: For 200 mL solution, mix 2 mL 1 M KCl, 400 μL
1 M MgCl 2 , 400 μL 1 M CaCl 2 , 0.2 g BSA, 78 mg 2-(N-morpholino) ethanesulfonic acid hydrate (MES), and 21.86 g mannitol (giving the following concentrations: 10 mM KCl, 2 mM
MgCl 2 , 2 mM CaCl 2 , 1 mg/mL BSA, 2 mM MES, 0.6 M
mannitol). Adjust pH to 5.5 with 1 M Tris base; typically, less
than 50 μL is required.
3. Solution BL (for leaves; 50 mL): 50 mL Solution A, 750 mg
Cellulysin (1.5% w/v), and 50 mg Pectolyase (0.1% w/v).
Prepare immediately prior to tissue harvest.
4. Solution BR (for roots; 50 mL): 50 mL Solution A, 750 mg
Cellulase RS (1.5% w/v), and 100 mg Macerozyme R10 (0.2%
w/v). Prepare immediately prior to tissue harvest.
5. 20.5% (w/v) sucrose dissolved in Solution A.
6. Supplies: BD Falcon™ or Sysmex 40, 70, 100 μm pore size
disposable mesh filters; disposable plastic transfer pipettes;
No. 22 scalpel; single-edged razor blades honed to a thickness
of 0.2 mm (we recommend VWR no. 55411-050). Kimwipes
paper tissues or similar. Plastic petri dishes (60 Â 15 cm).
Hemocytometer. BD Falcon™ 15 and 50 mL plastic centrifuge
tubes.
270
David W. Galbraith and Guiling Sun