3500. After acquisition is completed, linear regions are positioned around the various population distributions (2C, 4C,
etc.) of nuclei, to provide the mean or median amounts of
fluorescence and the coefficient of variation (CV) associated
with each peak.
11. Add a statistics panel, via the statistics tab on the acquisition
page, and customize this panel by right-clicking on it to access
the Statistics Settings. The display can be considerably simplified since we are interested only in the properties of the PI
fluorescence (the PerCP channel).
12. Initiate acquisition, using the slow flow rate, which provides
the lowest peak CVs. Acquisition is completed when sufficient
nuclei have passed through the instrument to provide welldefined populations of nuclei that maintain a consistent fluorescent signal over time. Operationally, this can be defined as
when the height of the highest peak of nuclei peak in the
uniparametric display reaches a preset value (200 is reasonable;
going beyond this number provides no significant advantage,
but simply slows down the overall rate of sample processing).
The gating is adjusted after accumulation to optimize the peak
profiles.
13. Save the files in FCS format for further analyses, and export
histograms and statistics values as needed.
Typical results for Arabidopsis are illustrated in Fig. 2.
Panel A illustrates a biparametric scatter plot of PI fluorescence
(detected by the PerCP channel) versus Side Scatter. The endoreduplicated nuclei form five distinct clusters, equally spaced across
the abscissa therefore representing 2C, 4C, 8C, and 16C nuclei.
Fig. 2 Analysis of nuclear genome sizes for Arabidopsis using the CytoFlex. (a). Biparametric analysis of
PI-fluorescence versus Side Scatter. Polygonal gate P1 surrounds the five clusters of nuclei, (b). Biparametric
analysis of PI-fluorescence versus time of staining. Nuclei exhibiting stable staining are enclosed by
rectangular gate P7. (c). Uniparametric histogram of PI fluorescence illustrating five cleanly separated
peaks of nuclei
Flow Cytometry and Sorting in Arabidopsis
277
etc.) of nuclei, to provide the mean or median amounts of
fluorescence and the coefficient of variation (CV) associated
with each peak.
11. Add a statistics panel, via the statistics tab on the acquisition
page, and customize this panel by right-clicking on it to access
the Statistics Settings. The display can be considerably simplified since we are interested only in the properties of the PI
fluorescence (the PerCP channel).
12. Initiate acquisition, using the slow flow rate, which provides
the lowest peak CVs. Acquisition is completed when sufficient
nuclei have passed through the instrument to provide welldefined populations of nuclei that maintain a consistent fluorescent signal over time. Operationally, this can be defined as
when the height of the highest peak of nuclei peak in the
uniparametric display reaches a preset value (200 is reasonable;
going beyond this number provides no significant advantage,
but simply slows down the overall rate of sample processing).
The gating is adjusted after accumulation to optimize the peak
profiles.
13. Save the files in FCS format for further analyses, and export
histograms and statistics values as needed.
Typical results for Arabidopsis are illustrated in Fig. 2.
Panel A illustrates a biparametric scatter plot of PI fluorescence
(detected by the PerCP channel) versus Side Scatter. The endoreduplicated nuclei form five distinct clusters, equally spaced across
the abscissa therefore representing 2C, 4C, 8C, and 16C nuclei.
Fig. 2 Analysis of nuclear genome sizes for Arabidopsis using the CytoFlex. (a). Biparametric analysis of
PI-fluorescence versus Side Scatter. Polygonal gate P1 surrounds the five clusters of nuclei, (b). Biparametric
analysis of PI-fluorescence versus time of staining. Nuclei exhibiting stable staining are enclosed by
rectangular gate P7. (c). Uniparametric histogram of PI fluorescence illustrating five cleanly separated
peaks of nuclei
Flow Cytometry and Sorting in Arabidopsis
277