variability associated with the unpredictable intracellular location of
the fluorescent nucleus within the protoplast, and the Gaussian
distribution of the focused laser illumination in the cytometer
across the flow stream within which the protoplast is hydrodynamically centered. As a result, CV values for the DNA contents of
(necessarily fixed) protoplasts are always larger than those of nuclei.
Coupled to the observation that it is not possible to produce
protoplasts from all species and organ types, whereas chopping to
produce homogenates containing nuclei is almost always feasible,
cytometric analysis of nuclei and not protoplasts is recommended
for all DNA-based measurements. Parenthetically, CVs of the
nuclear peaks of less than 2% can now be routinely achieved for
many major crop species using the latest generation of cytometers,
such as the CytoFLEX (see Note 1).
1.8 Dealing with Low
Amounts of RNA for
Expression Profiling
Modern flow sorters can routinely operate at sort rates of around
40,000 cells per second, but the rate of recovery of specific cells is
generally much lower. It is limited by the proportion of the labeled
cells within the source tissue, by the settings employed for the
operation of the sorter (whether in single or multi-droplet mode)
and, as previously discussed, by the size of the flow tip. The total
RNA content of eukaryotic cells is typically in the range of
1–100 pg. Global transcriptional profiling now almost exclusively
involves “Next” and subsequent “Generations” of high throughput DNA sequencing [132]. Amplification techniques are required
for studies integrating flow sorting with transcript analysis. Commercial kits have been developed that now extend to the single cell/
single nucleus level. We refer you to a previous review for details of
some of these methods [1], but in this review we now acknowledge
the increasing market penetration of commercial cDNA amplification/sequencing operations for transcriptional profiling, commenting as to how to integrate the contributions of these
companies to upstream manipulations using flow cytometry and
sorting. Finally, we describe how flow analysis and sorting can be
integrated with the 10Â Genomics Chromium Controller [133] to
provide input samples for agnostic determination of gene expression states.
1.9 Special Cases:
Flow Analysis and
Sorting of Pollen and
Sperm Cells
For many plant species, natural single cell suspensions are found
during male gametophyte development, most obviously in the
form of pollen. Pollen, being mechanically sturdy and frequently
autofluorescent, is well suited for flow cytometric analysis and
sorting [24, 128, 129]. Different species have characteristic pollen
diameters, and populations are frequently monodisperse. Successful
flow analysis and sorting of arabidopsis pollen and sperm cells
released from pollen homogenates led to the characterization of
gene expression within these distinctive cell types [1–3]. Promoter
sequences that specifically direct expression within these cells have
268
David W. Galbraith and Guiling Sun
the fluorescent nucleus within the protoplast, and the Gaussian
distribution of the focused laser illumination in the cytometer
across the flow stream within which the protoplast is hydrodynamically centered. As a result, CV values for the DNA contents of
(necessarily fixed) protoplasts are always larger than those of nuclei.
Coupled to the observation that it is not possible to produce
protoplasts from all species and organ types, whereas chopping to
produce homogenates containing nuclei is almost always feasible,
cytometric analysis of nuclei and not protoplasts is recommended
for all DNA-based measurements. Parenthetically, CVs of the
nuclear peaks of less than 2% can now be routinely achieved for
many major crop species using the latest generation of cytometers,
such as the CytoFLEX (see Note 1).
1.8 Dealing with Low
Amounts of RNA for
Expression Profiling
Modern flow sorters can routinely operate at sort rates of around
40,000 cells per second, but the rate of recovery of specific cells is
generally much lower. It is limited by the proportion of the labeled
cells within the source tissue, by the settings employed for the
operation of the sorter (whether in single or multi-droplet mode)
and, as previously discussed, by the size of the flow tip. The total
RNA content of eukaryotic cells is typically in the range of
1–100 pg. Global transcriptional profiling now almost exclusively
involves “Next” and subsequent “Generations” of high throughput DNA sequencing [132]. Amplification techniques are required
for studies integrating flow sorting with transcript analysis. Commercial kits have been developed that now extend to the single cell/
single nucleus level. We refer you to a previous review for details of
some of these methods [1], but in this review we now acknowledge
the increasing market penetration of commercial cDNA amplification/sequencing operations for transcriptional profiling, commenting as to how to integrate the contributions of these
companies to upstream manipulations using flow cytometry and
sorting. Finally, we describe how flow analysis and sorting can be
integrated with the 10Â Genomics Chromium Controller [133] to
provide input samples for agnostic determination of gene expression states.
1.9 Special Cases:
Flow Analysis and
Sorting of Pollen and
Sperm Cells
For many plant species, natural single cell suspensions are found
during male gametophyte development, most obviously in the
form of pollen. Pollen, being mechanically sturdy and frequently
autofluorescent, is well suited for flow cytometric analysis and
sorting [24, 128, 129]. Different species have characteristic pollen
diameters, and populations are frequently monodisperse. Successful
flow analysis and sorting of arabidopsis pollen and sperm cells
released from pollen homogenates led to the characterization of
gene expression within these distinctive cell types [1–3]. Promoter
sequences that specifically direct expression within these cells have
268
David W. Galbraith and Guiling Sun