histogram of fluorescence intensities. Acquire a polygonal or
amorphous gate on a dot-plot of SSC versus FL3(A) and establish the regions of identification for each ploidy category on a
uniparametric histogram, using the ProSort software. Calculate
the percentages of the nuclei in these ploidy categories using
Microsoft Excel.
4. For nuclei accumulating GFP, visualize the positions of the
desired nuclei through bivariate analysis of log green fluorescence (FL2A) versus log PI fluorescence (FL3(A)). Adjust a
rectangular or amorphous sort window to enclose the desired
nuclear population. Perform sorting in enrichment mode, with
1 or 2 droplets being sorted per positive event.
5. For analyzing and sorting nuclei stained with DAPI, follow the
same procedures indicated above for PI, except using FL1
(A) in the place of FL3(A). DAPI excitation will require the
405 nm laser.
3.3.3 Processing Sorted
Protoplasts and Nuclei
1. Validate instrument sort parameters by sorting 100 protoplasts
or nuclei onto a microscope slide, and counting these under a
microscope.
2. Sort protoplasts into 0.6 or 1.5 mL microcentrifuge tubes, or
into 96-well plates. For isolation of RNA from populations of
protoplasts, we sort into tubes prefilled with 3.5 volumes of
Lysis Buffer taken from the RNAqueous
®
-Micro Kit, assuming
one final volume of sorted protoplasts. For sorting single protoplasts, prefill the tubes with 5 μL of the Lysis Buffer
described in Subheading 3.5.2. Immediately shake the tubes
to ensure the sorted protoplast contacts the buffer as quickly as
possible.
3. Place the tubes on ice.
3.4 Flow Analysis of
Protoplasts and Nuclei
Expressing
Fluorescent Proteins
other than GFP
The same general principles apply as described above, with the one
exception of employing laser excitation and PMT detector filters
appropriate for the FP being used. On-line interactive websites
allowing optimization of optical filters for specific FPs can be
found at https://www.thermofisher.com/us/en/home/life-sci
ence/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.
html and http://www.bdbiosciences.com/research/multicolor/
spectrum_viewer/.
3.5 Amplification of
Targets from Sorted
Protoplasts and Nuclei
It is particularly important to rapidly denature the RNA samples
immediately following sorting. RNA is labile, and contamination
by ubiquitous RNases is difficult to avoid. Disposable gloves should
be worn, all aqueous solutions should be made using DEPCtreated diH 2 O, and glassware should be autoclaved.
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David W. Galbraith and Guiling Sun
amorphous gate on a dot-plot of SSC versus FL3(A) and establish the regions of identification for each ploidy category on a
uniparametric histogram, using the ProSort software. Calculate
the percentages of the nuclei in these ploidy categories using
Microsoft Excel.
4. For nuclei accumulating GFP, visualize the positions of the
desired nuclei through bivariate analysis of log green fluorescence (FL2A) versus log PI fluorescence (FL3(A)). Adjust a
rectangular or amorphous sort window to enclose the desired
nuclear population. Perform sorting in enrichment mode, with
1 or 2 droplets being sorted per positive event.
5. For analyzing and sorting nuclei stained with DAPI, follow the
same procedures indicated above for PI, except using FL1
(A) in the place of FL3(A). DAPI excitation will require the
405 nm laser.
3.3.3 Processing Sorted
Protoplasts and Nuclei
1. Validate instrument sort parameters by sorting 100 protoplasts
or nuclei onto a microscope slide, and counting these under a
microscope.
2. Sort protoplasts into 0.6 or 1.5 mL microcentrifuge tubes, or
into 96-well plates. For isolation of RNA from populations of
protoplasts, we sort into tubes prefilled with 3.5 volumes of
Lysis Buffer taken from the RNAqueous
®
-Micro Kit, assuming
one final volume of sorted protoplasts. For sorting single protoplasts, prefill the tubes with 5 μL of the Lysis Buffer
described in Subheading 3.5.2. Immediately shake the tubes
to ensure the sorted protoplast contacts the buffer as quickly as
possible.
3. Place the tubes on ice.
3.4 Flow Analysis of
Protoplasts and Nuclei
Expressing
Fluorescent Proteins
other than GFP
The same general principles apply as described above, with the one
exception of employing laser excitation and PMT detector filters
appropriate for the FP being used. On-line interactive websites
allowing optimization of optical filters for specific FPs can be
found at https://www.thermofisher.com/us/en/home/life-sci
ence/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.
html and http://www.bdbiosciences.com/research/multicolor/
spectrum_viewer/.
3.5 Amplification of
Targets from Sorted
Protoplasts and Nuclei
It is particularly important to rapidly denature the RNA samples
immediately following sorting. RNA is labile, and contamination
by ubiquitous RNases is difficult to avoid. Disposable gloves should
be worn, all aqueous solutions should be made using DEPCtreated diH 2 O, and glassware should be autoclaved.
284
David W. Galbraith and Guiling Sun