3.2 Preparation of
Plant Homogenates
and Flow Analysis of
Nuclei
1. Excise plant materials (organs or tissues) and, if necessary, wash
using diH 2 O. Blot dry. Transfer to a plastic petri dish
(60 Â 15 mm). The remaining procedures should be done on
ice and, ideally, in a walk-in cold room. Place the petri dish on a
prechilled ceramic tile embedded in an ice-filled plastic tray.
2. Add chopping buffer (2 mL per 0.5 g of fresh weight tissue is a
convenient proportion for these dishes). Chop tissues using a
new razor blade for 2–3 min.
3. Filter the homogenate through a 30 μm CellTrics
® disposable
filter.
4. Transfer an aliquot (0.5 mL) of the filtered homogenate to a
labeled tube. If to be stained using PI, add 2.5 μL of a 10 mg/
mL solution of DNAse-free RNAse A. Incubate on ice for
10 min. Add PI to a final concentration of 50 μg/mL. If to
be stained with DAPI, add the aliquot to a tube containing
DAPI to provide a final concentration of 20 μg/mL.
5. Incubate the stained samples on ice in darkness for 20 min prior
to flow cytometric analysis.
3.2.1 Instrument Settings
for Flow Cytometric
Analyses of Nuclei
The flow cytometer should be configured to efficiently excite and
detect fluorescence produced by the propidium iodide-DNA or
DAPI-DNA complexes found in stained nuclei. The general configuration involves the following elements for all cytometers:
(a) Optimal excitation for PI occurs over the range of
475–575 nm (maximum at 535 nm), so can be achieved using
laser lines at 488 nm or 561 nm. Emission occurs over
580–700 nm (maximum 617 nm), so dichroics and barrier filter
combinations should be chosen that capture light across this region
and that are compatible with the laser excitation line. For DAPI,
excitation is optimal around 355 nm, but is still possible using the
405 nm line, and the emission maximum is around 460 nm. Since
cellular homogenates contain a large proportion of irrelevant
(non-nuclear) particles, some of which may be autofluorescent
(cf. chloroplasts), and some nonfluorescent (mitochondria, endomembrane vesicles, etc.), but all of which produce scatter signals, it
is recommended that triggering be done on the fluorescence signals
(FL1(A) or FL3(A), depending on the fluorochrome), rather than
on forward-angle light scatter. Then the threshold detection level
of the fluorescence channel should be adjusted upward to eliminate
all irrelevant signals, at the same time taking care not to exclude the
nuclear peaks of interest, in particular the lowest (2C) peak. For
Arabidopsis homogenates stained with PI, biparametric plots of
fluorescence emission spanning 545–625 nm (orange) versus
>670 nm (red) can be helpful in identifying the position of the
nuclei, since these will all fall along a discrete linear region-ofinterest corresponding to the distribution of the PI-DNA signal
274
David W. Galbraith and Guiling Sun
Plant Homogenates
and Flow Analysis of
Nuclei
1. Excise plant materials (organs or tissues) and, if necessary, wash
using diH 2 O. Blot dry. Transfer to a plastic petri dish
(60 Â 15 mm). The remaining procedures should be done on
ice and, ideally, in a walk-in cold room. Place the petri dish on a
prechilled ceramic tile embedded in an ice-filled plastic tray.
2. Add chopping buffer (2 mL per 0.5 g of fresh weight tissue is a
convenient proportion for these dishes). Chop tissues using a
new razor blade for 2–3 min.
3. Filter the homogenate through a 30 μm CellTrics
® disposable
filter.
4. Transfer an aliquot (0.5 mL) of the filtered homogenate to a
labeled tube. If to be stained using PI, add 2.5 μL of a 10 mg/
mL solution of DNAse-free RNAse A. Incubate on ice for
10 min. Add PI to a final concentration of 50 μg/mL. If to
be stained with DAPI, add the aliquot to a tube containing
DAPI to provide a final concentration of 20 μg/mL.
5. Incubate the stained samples on ice in darkness for 20 min prior
to flow cytometric analysis.
3.2.1 Instrument Settings
for Flow Cytometric
Analyses of Nuclei
The flow cytometer should be configured to efficiently excite and
detect fluorescence produced by the propidium iodide-DNA or
DAPI-DNA complexes found in stained nuclei. The general configuration involves the following elements for all cytometers:
(a) Optimal excitation for PI occurs over the range of
475–575 nm (maximum at 535 nm), so can be achieved using
laser lines at 488 nm or 561 nm. Emission occurs over
580–700 nm (maximum 617 nm), so dichroics and barrier filter
combinations should be chosen that capture light across this region
and that are compatible with the laser excitation line. For DAPI,
excitation is optimal around 355 nm, but is still possible using the
405 nm line, and the emission maximum is around 460 nm. Since
cellular homogenates contain a large proportion of irrelevant
(non-nuclear) particles, some of which may be autofluorescent
(cf. chloroplasts), and some nonfluorescent (mitochondria, endomembrane vesicles, etc.), but all of which produce scatter signals, it
is recommended that triggering be done on the fluorescence signals
(FL1(A) or FL3(A), depending on the fluorochrome), rather than
on forward-angle light scatter. Then the threshold detection level
of the fluorescence channel should be adjusted upward to eliminate
all irrelevant signals, at the same time taking care not to exclude the
nuclear peaks of interest, in particular the lowest (2C) peak. For
Arabidopsis homogenates stained with PI, biparametric plots of
fluorescence emission spanning 545–625 nm (orange) versus
>670 nm (red) can be helpful in identifying the position of the
nuclei, since these will all fall along a discrete linear region-ofinterest corresponding to the distribution of the PI-DNA signal
274
David W. Galbraith and Guiling Sun