been identified [134–136]. Regulated FP expression can be used to
subdivide microspores, pollen, and sperm cells according to function and allow flow analysis and sorting to purify sperm cells,
vegetative nuclei, and microspores [3]. In this case, sperm cells
and vegetative nuclei were specifically identified by expression of
GFP and RFP, and microspores identified based on their size and
autofluorescence properties. Misra et al. [137] have described
methods for the characterization of the transcriptomes of single
sperm cells isolated from pollen tubes. Recently, Luria et al. [5]
directly examined pollen fitness using flow cytometry and sorting.
Dichlorofluorescein diacetate staining was used to discriminate
between metabolically active, viable pollen, and less active and
dead cells, corresponding to bimodal distributions of fluorescence
emission. Pollen, flow sorted from these peaks, is capable of germination in vitro, the rate of germination reflecting the recorded level
of metabolic activity, and being sensitive to stress treatments. This
implies that flow cytometry and sorting can be used as both a stress
diagnostic and as a means to enrich viable pollen. In other work,
Yelina et al. [138] devised a three-color assay to rapidly screen
low-frequency crossover events within large populations of pollen
expressing eCFP, dsRed, and eYFP. This approach has facilitated
progress in understanding the control of meiotic recombination
[139, 140].
Finally, it can be noted that the Union Biometrica instruments
can be configured to analyze and sort pollen, further details being
found at their website.
1.10 Major
Equipment
Not all flow cytometers are cell sorters. Cell sorters always cost
more than flow cytometers, and sometimes are considerably more
expensive. Cell sorters are generally found within core facilities and,
being more complicated than flow cytometers, are run by dedicated
operators on a fee-for-service basis. Most of the current models of
commercial flow sorters can be equipped with large flow tips,
100 μm being standard, and this is usually sufficient for sorting
plant protoplasts, and certainly for those from arabidopsis. We have
had positive, first-hand experiences with flow sorters from Beckman
Coulter, Becton-Dickinson, Thermo-Fisher, BioRad, and SONY.
Flow cytometers can cost as little as $50,000, and there is commercial interest in driving this price point further down, particularly for
portable instruments. Again, all currently manufactured flow cytometers as well as the recently emerging spectral analyzers appear
suitable for use with arabidopsis.
Given the decreasing price of flow cytometers, their improved
performance, and the incorporation of automation into setup and
troubleshooting procedures, these instruments are increasingly
appearing in end-user laboratories. Strongly recommended is the
availability of a good quality fluorescence microscope in close proximity to the flow cytometer/cell sorter. This is particularly important for verification of the presence of fluorescence labeling within
Flow Cytometry and Sorting in Arabidopsis
269
subdivide microspores, pollen, and sperm cells according to function and allow flow analysis and sorting to purify sperm cells,
vegetative nuclei, and microspores [3]. In this case, sperm cells
and vegetative nuclei were specifically identified by expression of
GFP and RFP, and microspores identified based on their size and
autofluorescence properties. Misra et al. [137] have described
methods for the characterization of the transcriptomes of single
sperm cells isolated from pollen tubes. Recently, Luria et al. [5]
directly examined pollen fitness using flow cytometry and sorting.
Dichlorofluorescein diacetate staining was used to discriminate
between metabolically active, viable pollen, and less active and
dead cells, corresponding to bimodal distributions of fluorescence
emission. Pollen, flow sorted from these peaks, is capable of germination in vitro, the rate of germination reflecting the recorded level
of metabolic activity, and being sensitive to stress treatments. This
implies that flow cytometry and sorting can be used as both a stress
diagnostic and as a means to enrich viable pollen. In other work,
Yelina et al. [138] devised a three-color assay to rapidly screen
low-frequency crossover events within large populations of pollen
expressing eCFP, dsRed, and eYFP. This approach has facilitated
progress in understanding the control of meiotic recombination
[139, 140].
Finally, it can be noted that the Union Biometrica instruments
can be configured to analyze and sort pollen, further details being
found at their website.
1.10 Major
Equipment
Not all flow cytometers are cell sorters. Cell sorters always cost
more than flow cytometers, and sometimes are considerably more
expensive. Cell sorters are generally found within core facilities and,
being more complicated than flow cytometers, are run by dedicated
operators on a fee-for-service basis. Most of the current models of
commercial flow sorters can be equipped with large flow tips,
100 μm being standard, and this is usually sufficient for sorting
plant protoplasts, and certainly for those from arabidopsis. We have
had positive, first-hand experiences with flow sorters from Beckman
Coulter, Becton-Dickinson, Thermo-Fisher, BioRad, and SONY.
Flow cytometers can cost as little as $50,000, and there is commercial interest in driving this price point further down, particularly for
portable instruments. Again, all currently manufactured flow cytometers as well as the recently emerging spectral analyzers appear
suitable for use with arabidopsis.
Given the decreasing price of flow cytometers, their improved
performance, and the incorporation of automation into setup and
troubleshooting procedures, these instruments are increasingly
appearing in end-user laboratories. Strongly recommended is the
availability of a good quality fluorescence microscope in close proximity to the flow cytometer/cell sorter. This is particularly important for verification of the presence of fluorescence labeling within
Flow Cytometry and Sorting in Arabidopsis
269