fluorescence spectra for each individual nucleus. After deconvolution using the library spectra for GFP and PI-DNA, along with the
spectra obtained from non-stained and non-transgenic controls,
identification of GFP-accumulating and non-accumulating nuclei
is easily achieved (Panel B). There are little differences in the
proportions of 2C, 4C, 8C, etc., nuclei within these two populations (Panels C, D). In that the original transgenic plants were
produced in 2006, and have been through multiple selfed generations since that time, we conclude that stable constitutive GFP
expression and accumulation within the nuclei has been achieved.
3.3 Flow Sorting of
Protoplasts, Pollen and
Nuclei
A detailed description of the setting up and operation of the
Beckman-Coulter Mo-Flo has been provided previously [1]. For
sorting with the BioRad S3, we routinely employ a 100 μm flow tip,
and purity sorting mode. In this mode, the target droplet is sorted
only if there is no contaminating particle in the droplet before
and/or after the target droplet.
1. Before or immediately after switching on the cytometer, but
before starting analyses, check the waste and sheath fluid bottles, and empty or refill them, as required. Start the ProSort
software and select startup under the Setup and Maintenance
tab. After warm up, run the QC program to prepare the
instrument for use.
2. Establish conditions for sorting using standard indestructible
fluorescent particles that approximate the size of the protoplasts or nuclei that are being sorted. 10 μm Flow-Check™
Fluorospheres are appropriate for sorting nuclei. For the larger
protoplasts, larger fluorescent particles are recommended, such
as pollen. Particles as large as 50% of the diameter of the flow
tip can be successfully sorted and at high recovery rates
[129]. In the interest of establishing stable sorting conditions
with minimal disturbance to protoplast integrity and recovery,
it is often recommended that the flow tip diameter be at least
three-fold larger than the average diameter of the protoplasts.
This conflicts with issues concerning the sample volumes generated during sorting, since droplet volume scales dramatically
with increases in tip diameter. With the S3, we routinely employ
a sheath pressure of 30 PSI, a typical droplet drive frequency of
38–42 kHz, and a drop delay value of 33–35. These parameters
are automatically established by the instrument.
3. The optical configuration of the S3 is as follows: For a two-laser
(488 nm/561 nm) system, the standard filters are FL1
[525/30 nm], FL2 [586/25 nm], FL3 [615/25 nm], and
FL4 [655 nm LP]. For a three-laser system (405/488/
561 nm), the filters are FL1 [447/60 nm], FL2
[525/30 nm], FL3 [586/25 nm], and FL4 [600 nm LP].
Laser illumination in all cases is co-linear, and for most applications, all lasers are switched on and produce 100 mW.
280
David W. Galbraith and Guiling Sun
spectra obtained from non-stained and non-transgenic controls,
identification of GFP-accumulating and non-accumulating nuclei
is easily achieved (Panel B). There are little differences in the
proportions of 2C, 4C, 8C, etc., nuclei within these two populations (Panels C, D). In that the original transgenic plants were
produced in 2006, and have been through multiple selfed generations since that time, we conclude that stable constitutive GFP
expression and accumulation within the nuclei has been achieved.
3.3 Flow Sorting of
Protoplasts, Pollen and
Nuclei
A detailed description of the setting up and operation of the
Beckman-Coulter Mo-Flo has been provided previously [1]. For
sorting with the BioRad S3, we routinely employ a 100 μm flow tip,
and purity sorting mode. In this mode, the target droplet is sorted
only if there is no contaminating particle in the droplet before
and/or after the target droplet.
1. Before or immediately after switching on the cytometer, but
before starting analyses, check the waste and sheath fluid bottles, and empty or refill them, as required. Start the ProSort
software and select startup under the Setup and Maintenance
tab. After warm up, run the QC program to prepare the
instrument for use.
2. Establish conditions for sorting using standard indestructible
fluorescent particles that approximate the size of the protoplasts or nuclei that are being sorted. 10 μm Flow-Check™
Fluorospheres are appropriate for sorting nuclei. For the larger
protoplasts, larger fluorescent particles are recommended, such
as pollen. Particles as large as 50% of the diameter of the flow
tip can be successfully sorted and at high recovery rates
[129]. In the interest of establishing stable sorting conditions
with minimal disturbance to protoplast integrity and recovery,
it is often recommended that the flow tip diameter be at least
three-fold larger than the average diameter of the protoplasts.
This conflicts with issues concerning the sample volumes generated during sorting, since droplet volume scales dramatically
with increases in tip diameter. With the S3, we routinely employ
a sheath pressure of 30 PSI, a typical droplet drive frequency of
38–42 kHz, and a drop delay value of 33–35. These parameters
are automatically established by the instrument.
3. The optical configuration of the S3 is as follows: For a two-laser
(488 nm/561 nm) system, the standard filters are FL1
[525/30 nm], FL2 [586/25 nm], FL3 [615/25 nm], and
FL4 [655 nm LP]. For a three-laser system (405/488/
561 nm), the filters are FL1 [447/60 nm], FL2
[525/30 nm], FL3 [586/25 nm], and FL4 [600 nm LP].
Laser illumination in all cases is co-linear, and for most applications, all lasers are switched on and produce 100 mW.
280
David W. Galbraith and Guiling Sun