state lasers: 405 nm, 80 mW; 488 nm, 50 mW; 561 nm,
30 mW; and 638 nm, 50 mW.
It should be noted that the results require acquisition using
only the blue (488 nm) laser and a single fluorescence channel
(in this case, named “PerCP”) that collects light centered at
690 nm with a 50 nm spectral bandwidth.
5. Open the “New Experiment” dialog box in the File menu.
Choose a location for the experiment file.
6. Open the Detector Configuration window and verify that software (SW) and hardware (HW) configurations match.
7. Under the Settings tab, select “Set Channel” and de-select the
channels not in use. This is since we only require excitation
from one laser, and detection only from light scatter and the
one-color PI fluorescence (using the PerCP channel). Deselecting channels allows more total events to be acquired. One
disadvantage of this deselection is that further exploration of
endogenous fluorophores (chloroplasts, etc.) in relationship to
the nuclei is not possible.
8. Open the “Acq. Settings” window. Select the “Gain” tab.
Adjust the gain settings to correspond to FS 80, SSC,
20, PerCP fluorescence 25.
9. Select the “Threshold” tab. Adjust the settings to manual and
10,000. Note that triggering is done on the PerCP fluorescence channel, rather than on FS or SS as would be done for
cell suspensions. If needed, adjust upward the threshold level to
exclude from consideration more of the objects having lower
fluorescence than the PI-stained nuclei. This threshold level is a
function of the gain setting chosen for the PerCP channel, and
the size of the genome being analyzed, but the suggested
values should be appropriate for Arabidopsis as long as the
instrument QC protocol is followed on start-up.
10. Close the “Threshold tab.” Create one single parameter histogram and two bivariate dot plots in the experiment space. The
first bivariate dot plot will be labeled as PerCP channel versus
Side Scatter, with the following properties: X-axis PerCP log
scale, minimum 10,000, maximum 16,500,000. Y-axis SSC
log scale, minimum 160, maximum 8,500,000. A polygonal
gate (P1) is drawn as indicated in Fig. X. The second bivariate
plot is of the PerCP-A channel versus Time, with the following
properties: X-axis Time linear scale, minimum 0, maximum
1,600,000. Y-axis PerCP log scale, minimum 195,000, maximum 16,750,000. A rectangular gate (P7) is drawn as shown
in Fig. X. The single parameter histogram is of the PerCP-A
Channel versus Count, gated on P1, with the following properties: X-axis PerCP log scale, minimum 10,000, maximum
16,500,000. Y-axis Count linear scale, minimum 0, maximum
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David W. Galbraith and Guiling Sun
30 mW; and 638 nm, 50 mW.
It should be noted that the results require acquisition using
only the blue (488 nm) laser and a single fluorescence channel
(in this case, named “PerCP”) that collects light centered at
690 nm with a 50 nm spectral bandwidth.
5. Open the “New Experiment” dialog box in the File menu.
Choose a location for the experiment file.
6. Open the Detector Configuration window and verify that software (SW) and hardware (HW) configurations match.
7. Under the Settings tab, select “Set Channel” and de-select the
channels not in use. This is since we only require excitation
from one laser, and detection only from light scatter and the
one-color PI fluorescence (using the PerCP channel). Deselecting channels allows more total events to be acquired. One
disadvantage of this deselection is that further exploration of
endogenous fluorophores (chloroplasts, etc.) in relationship to
the nuclei is not possible.
8. Open the “Acq. Settings” window. Select the “Gain” tab.
Adjust the gain settings to correspond to FS 80, SSC,
20, PerCP fluorescence 25.
9. Select the “Threshold” tab. Adjust the settings to manual and
10,000. Note that triggering is done on the PerCP fluorescence channel, rather than on FS or SS as would be done for
cell suspensions. If needed, adjust upward the threshold level to
exclude from consideration more of the objects having lower
fluorescence than the PI-stained nuclei. This threshold level is a
function of the gain setting chosen for the PerCP channel, and
the size of the genome being analyzed, but the suggested
values should be appropriate for Arabidopsis as long as the
instrument QC protocol is followed on start-up.
10. Close the “Threshold tab.” Create one single parameter histogram and two bivariate dot plots in the experiment space. The
first bivariate dot plot will be labeled as PerCP channel versus
Side Scatter, with the following properties: X-axis PerCP log
scale, minimum 10,000, maximum 16,500,000. Y-axis SSC
log scale, minimum 160, maximum 8,500,000. A polygonal
gate (P1) is drawn as indicated in Fig. X. The second bivariate
plot is of the PerCP-A channel versus Time, with the following
properties: X-axis Time linear scale, minimum 0, maximum
1,600,000. Y-axis PerCP log scale, minimum 195,000, maximum 16,750,000. A rectangular gate (P7) is drawn as shown
in Fig. X. The single parameter histogram is of the PerCP-A
Channel versus Count, gated on P1, with the following properties: X-axis PerCP log scale, minimum 10,000, maximum
16,500,000. Y-axis Count linear scale, minimum 0, maximum
276
David W. Galbraith and Guiling Sun