2.3.4 Liquid Cultured
Plant Cells
Cell pellets should be compact and dense. Hence, cells grown in
liquid culture must be concentrated, usually by centrifugation
before freezing. The sample should resemble a thick paste.
1. Spin down a 1.5 mL aliquot of the liquid culture using a
tabletop centrifuge for 1 min at 1000 Â g.
2. Discard the supernatant and resuspend in a small volume of
40% dextran.
3. Mount the bottom part of the brass carrier onto the Bal-tec
HPM 010 freezing rod.
4. With a toothpick transfer the cells into the freezing carrier and
add a drop of 40% dextran.
2.3.5 Sample Position
Strategy
How the sample is positioned in the carrier will determine the
cutting angle of the tissue (transversal or longitudinal) and can
make the trimming process easier and more efficient.
Because each project and sample have their own requirements,
here we will detail the advantages and disadvantages of two types of
carriers used in our laboratory.
1. 200 μm brass carrier (used for PSB-D cell pellets)
In this system, the sample + cryoprotectant filler are laid in
the bottom part of the carrier to form a prominent bubble,
which is then enclosed by the concave top of the carrier
(Fig. 3a). This system allows rapid trimming because the sample is more prominent; however, vitrification is only optimal in
the edges of the sample, with the deeper parts usually forming
hexagonal ice. This type of holder is appropriate for dense
pellets of cells, where direction and area do not matter since
the cells are randomly oriented in the pellet.
2. 100 μm type A gold-plated copper carrier (used for Arabidopsis root tips).
In this carrier, the sample + cryoprotectant filler forms a flat
film within the rim of the carrier (Fig. 3b). This provides the
advantage of homogenous vitrification throughout the thin
sample. The disadvantage is that the carrier needs to be
trimmed from the side until the sample is reached (Fig. 3b).
This system is appropriate for tissues less than 100 μm thick
that require sectioning in a particular orientation.
After positioning the sample in the desired way inside the
carrier, place the lid of the carrier on top of the sample, and
secure it on the freezing rod and subsequently put it into the
slot of the Bal-tec HPM 010 and perform HPF. From this
point on, the samples should never be taken out of liquid
nitrogen.
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Mohammed Kaplan et al.
Plant Cells
Cell pellets should be compact and dense. Hence, cells grown in
liquid culture must be concentrated, usually by centrifugation
before freezing. The sample should resemble a thick paste.
1. Spin down a 1.5 mL aliquot of the liquid culture using a
tabletop centrifuge for 1 min at 1000 Â g.
2. Discard the supernatant and resuspend in a small volume of
40% dextran.
3. Mount the bottom part of the brass carrier onto the Bal-tec
HPM 010 freezing rod.
4. With a toothpick transfer the cells into the freezing carrier and
add a drop of 40% dextran.
2.3.5 Sample Position
Strategy
How the sample is positioned in the carrier will determine the
cutting angle of the tissue (transversal or longitudinal) and can
make the trimming process easier and more efficient.
Because each project and sample have their own requirements,
here we will detail the advantages and disadvantages of two types of
carriers used in our laboratory.
1. 200 μm brass carrier (used for PSB-D cell pellets)
In this system, the sample + cryoprotectant filler are laid in
the bottom part of the carrier to form a prominent bubble,
which is then enclosed by the concave top of the carrier
(Fig. 3a). This system allows rapid trimming because the sample is more prominent; however, vitrification is only optimal in
the edges of the sample, with the deeper parts usually forming
hexagonal ice. This type of holder is appropriate for dense
pellets of cells, where direction and area do not matter since
the cells are randomly oriented in the pellet.
2. 100 μm type A gold-plated copper carrier (used for Arabidopsis root tips).
In this carrier, the sample + cryoprotectant filler forms a flat
film within the rim of the carrier (Fig. 3b). This provides the
advantage of homogenous vitrification throughout the thin
sample. The disadvantage is that the carrier needs to be
trimmed from the side until the sample is reached (Fig. 3b).
This system is appropriate for tissues less than 100 μm thick
that require sectioning in a particular orientation.
After positioning the sample in the desired way inside the
carrier, place the lid of the carrier on top of the sample, and
secure it on the freezing rod and subsequently put it into the
slot of the Bal-tec HPM 010 and perform HPF. From this
point on, the samples should never be taken out of liquid
nitrogen.
94
Mohammed Kaplan et al.