Gate P1serves to isolate the nuclei for display over time in Panel
B. In this panel, the biparametric scatter plot of fluorescence intensity versus time indicates stability shortly after addition of the PI
stain. A second gate (P1) can be used to examine only those nuclei
whose fluorescence is stable. Panel C illustrates a uniparametric
histogram of PI fluorescence, gated on P1. The discrete peaks of
fluorescence corresponding to the 2C, 4C, 8C, and 16C nuclei are
clearly and cleanly separated, with excellent CV values (P2 ¼ 1.77%,
P3 ¼ 1.68%, P4 ¼ 1.39%, P5 ¼ 1.32%).
3.2.2 Instrument Settings
for Spectral Analysis of
Nuclei using the SA3800
1. Prior to startup, verify that there is sufficient sheath fluid in the
sheath fluid container (particle free water) and that the waste
container is empty.
2. Start the SA3800 software, Prime the fluidics, then run the QC
software using the SONY AlignCheck beads. SONY 8-Peak
beads can also be used to provide additional sensitivity and
verification of linearity.
3. Create a new experiment by either using the library of stored
fluorochromes, an existing template, or a blank template. Select
Normal or Standardization mode. Define your tubes/plates.
Set up scatterplots plots and histograms to view your data.
4. For cell cycle analysis, a dual-region gating strategy can be
utilized, involving an initial region within the FSC-A versus
SSC-A scatterplot to eliminate nonspecific debris, and a second
region within the ECD-A versus ECD-H scatterplot to eliminate doublets; both regions are used to gate the final uniparametric DNA content histograms (ECD-A).
5. For plant homogenate analysis, no fluorochrome patterns
(“colors”) need to be preset, since the data can be first accumulated and the colors defined later from these. The spectra of
the unstained versus PI-stained wildtype control and transgenic
plants (four samples) are acquired separately to provide appropriate background measurements.
6. First, a control sample stained with PI is analyzed, adjusting the
PMT voltage to position the nuclei on scale. A negative
(unstained) control is then run in order to define and subtract
any autofluorescence. Subsequent transgenic samples expressing nuclear GFP are run along with a negative control as
necessary (if the PMT voltages are different from those of the
original sample). The fluorochrome patterns (“colors”) are
then saved into the library.
7. Subsequent samples are then run using the colors from the
library, the only required control being the negative for each
homogenate type. These negative controls allow subtraction of
any cellular autofluorescence from the GFP and PI fluorescence
signals.
278
David W. Galbraith and Guiling Sun
B. In this panel, the biparametric scatter plot of fluorescence intensity versus time indicates stability shortly after addition of the PI
stain. A second gate (P1) can be used to examine only those nuclei
whose fluorescence is stable. Panel C illustrates a uniparametric
histogram of PI fluorescence, gated on P1. The discrete peaks of
fluorescence corresponding to the 2C, 4C, 8C, and 16C nuclei are
clearly and cleanly separated, with excellent CV values (P2 ¼ 1.77%,
P3 ¼ 1.68%, P4 ¼ 1.39%, P5 ¼ 1.32%).
3.2.2 Instrument Settings
for Spectral Analysis of
Nuclei using the SA3800
1. Prior to startup, verify that there is sufficient sheath fluid in the
sheath fluid container (particle free water) and that the waste
container is empty.
2. Start the SA3800 software, Prime the fluidics, then run the QC
software using the SONY AlignCheck beads. SONY 8-Peak
beads can also be used to provide additional sensitivity and
verification of linearity.
3. Create a new experiment by either using the library of stored
fluorochromes, an existing template, or a blank template. Select
Normal or Standardization mode. Define your tubes/plates.
Set up scatterplots plots and histograms to view your data.
4. For cell cycle analysis, a dual-region gating strategy can be
utilized, involving an initial region within the FSC-A versus
SSC-A scatterplot to eliminate nonspecific debris, and a second
region within the ECD-A versus ECD-H scatterplot to eliminate doublets; both regions are used to gate the final uniparametric DNA content histograms (ECD-A).
5. For plant homogenate analysis, no fluorochrome patterns
(“colors”) need to be preset, since the data can be first accumulated and the colors defined later from these. The spectra of
the unstained versus PI-stained wildtype control and transgenic
plants (four samples) are acquired separately to provide appropriate background measurements.
6. First, a control sample stained with PI is analyzed, adjusting the
PMT voltage to position the nuclei on scale. A negative
(unstained) control is then run in order to define and subtract
any autofluorescence. Subsequent transgenic samples expressing nuclear GFP are run along with a negative control as
necessary (if the PMT voltages are different from those of the
original sample). The fluorochrome patterns (“colors”) are
then saved into the library.
7. Subsequent samples are then run using the colors from the
library, the only required control being the negative for each
homogenate type. These negative controls allow subtraction of
any cellular autofluorescence from the GFP and PI fluorescence
signals.
278
David W. Galbraith and Guiling Sun