ChemoMetec NC-3000 - Manual (Page 47)

991-3003 Rev. 1.0 August 2013 47
3. In Plot Manager, open a new scatter plot by clicking on the scatter plot icon on the left-hand
side of the data row.
4. Double-click the small scatter plot to open the large scatter plot in editing mode. Change the x-axis
to the color of the fluorophore and the y-axis to the color of the neighboring channel. The axis
settings for both axes should be ‘Linear’ and 'Intensity'. Click 'OK'. Tip: Change the y-axis range so
that the cell population is centered on the y-axis.
5. If there is spillover of fluorescence between the channels the cell population will be placed
diagonally across the graph area. If there is no spillover, the cell population will be placed
horizontally across the graph area.
6. Open the compensation matrix in Plot Manager by clicking on the icon on the left-hand side of
the data row.
7. Enter an estimated compensation factor into the matrix box associated with the two channels of
interest and click 'Preview'. Inspect the cell population to ascertain that cell population is spread
horizontally across the graph area. If the compensation factor is incorrect, click 'Roll back' enter a
new value and inspect the cell population again. Repeat this procedure until the correct
compensation factor is ascertained and click 'Apply'.
8. Repeat steps 1-7 for each individual fluorophore.
9. If multiple samples have been analyzed, enter the row number of the sample with the corrected
compensation factor into the 'Master Row' box situated in the top left-hand corner of Plot Manager
and click 'Apply'. This will replicate the compensation in all other samples selected in Plot Manager.
Compensation matrix adjusted for
0.8% signal spillover from the GFP
into the orange channel.
Scatter plot showing GFP fluorescence
in the green and orange channel after
compensation. Note that there is no
increase in orange fluorescence as
green fluorescence increases. Scatter plot of GFP expressing cells
showing spillover of green fluorescence
into the orange channel. An increase in
orange fluorescence is seen as green
fluorescence increases.