homogenates), since cytometry requires individual interrogation of
the fluorescence and light-scatter properties of the particles as they
pass through laser illumination. Thus, methods are required to
produce these sample suspensions from plants. With the important
exception of pollen and sperm cells, which are natural single cell
suspensions that can be directly analyzed using flow cytometry and
cell sorting [2–5], conversion of multicellular tissues to single cells
requires protoplast production via removal of the cell wall
[6]. Removing the cell wall means that protoplasts are extremely
fragile, and since plant cells typically are larger than the mammalian
cells, around which flow cytometry and cell sorting instruments
were originally designed, this provides additional complications.
Protoplast production also represents a significant perturbation to
their cellular physiology, and this will impact interpretation of any
subsequent results. Since not all cell and tissue types or plant species
are amenable to protoplast production, this further limits this
strategy [6].
An alternative approach for applying flow cytometry to the
multicellular sporophyte bypasses the requirement for single cells
through analyzing of their subcellular contents following homogenization [7]. This approach is unusual to the shared resource
cytometry core, since the objects of interest are not the majority
of the objects being analyzed (as is the case for flow cytometric
analysis of cells or protoplasts). In contrast, analysis of homogenates represents a search for an extreme minority of objects of
interest within a vast sea of irrelevant debris; this significant technical complication is not always recognized.
This chapter provides protocols for employing flow cytometry
and sorting with Arabidopsis thaliana, covering issues related to
working both with protoplasts (including dealing with large particles whilst maintaining viability) and with nuclei (including identification of minor subpopulations, and dealing with
autofluorescence). The protocols also provide details of working
with transgenic organisms expressing Fluorescent Proteins (FPs),
and suggests avenues for exploration using novel FP variants. They
indicate how flow analysis and sorting can be integrated with
agnostic sampling of gene expression states. Tips for successful
operation of current cytometric instrumentation are included.
Our laboratories have worked with a number of different flow
cytometers and cell sorters, including the Coulter EPICS and
Elite series, the Beckman-Coulter MoFlo and CytoFLEX, the Becton Dickinson FACScan, FACSCalibur, LSR II, and Accuri C6
instruments, the LifeTechnologies Attune, and the BioRad S3.
The described methods are generally applicable to other flow cytometers and sorters, and broadly to plant species beyond Arabidopsis. In this chapter, we particularly include new information
concerning the use, with Arabidopsis, of the BioRad S3 sorter,
the CytoFLEX cytometer, and the SONY SH800 spectral analyzer.
256
David W. Galbraith and Guiling Sun
the fluorescence and light-scatter properties of the particles as they
pass through laser illumination. Thus, methods are required to
produce these sample suspensions from plants. With the important
exception of pollen and sperm cells, which are natural single cell
suspensions that can be directly analyzed using flow cytometry and
cell sorting [2–5], conversion of multicellular tissues to single cells
requires protoplast production via removal of the cell wall
[6]. Removing the cell wall means that protoplasts are extremely
fragile, and since plant cells typically are larger than the mammalian
cells, around which flow cytometry and cell sorting instruments
were originally designed, this provides additional complications.
Protoplast production also represents a significant perturbation to
their cellular physiology, and this will impact interpretation of any
subsequent results. Since not all cell and tissue types or plant species
are amenable to protoplast production, this further limits this
strategy [6].
An alternative approach for applying flow cytometry to the
multicellular sporophyte bypasses the requirement for single cells
through analyzing of their subcellular contents following homogenization [7]. This approach is unusual to the shared resource
cytometry core, since the objects of interest are not the majority
of the objects being analyzed (as is the case for flow cytometric
analysis of cells or protoplasts). In contrast, analysis of homogenates represents a search for an extreme minority of objects of
interest within a vast sea of irrelevant debris; this significant technical complication is not always recognized.
This chapter provides protocols for employing flow cytometry
and sorting with Arabidopsis thaliana, covering issues related to
working both with protoplasts (including dealing with large particles whilst maintaining viability) and with nuclei (including identification of minor subpopulations, and dealing with
autofluorescence). The protocols also provide details of working
with transgenic organisms expressing Fluorescent Proteins (FPs),
and suggests avenues for exploration using novel FP variants. They
indicate how flow analysis and sorting can be integrated with
agnostic sampling of gene expression states. Tips for successful
operation of current cytometric instrumentation are included.
Our laboratories have worked with a number of different flow
cytometers and cell sorters, including the Coulter EPICS and
Elite series, the Beckman-Coulter MoFlo and CytoFLEX, the Becton Dickinson FACScan, FACSCalibur, LSR II, and Accuri C6
instruments, the LifeTechnologies Attune, and the BioRad S3.
The described methods are generally applicable to other flow cytometers and sorters, and broadly to plant species beyond Arabidopsis. In this chapter, we particularly include new information
concerning the use, with Arabidopsis, of the BioRad S3 sorter,
the CytoFLEX cytometer, and the SONY SH800 spectral analyzer.
256
David W. Galbraith and Guiling Sun