For this tutorial, we will be using a layout file that has 2D density plots with defined gates from seven data files, each having different amounts of CD64 antibody. At anytime if you would like to skip ahead to the Create a Custom Data Table section the steps from the tutorial have been completed in a new layout for the next section.

The following layout should appear as shown in Figure T17.1.

 

 

In the upper left corner of the layout is a dot plot of Side Scatter versus Forward Scatter with a scatter gate, drawn in red. The Scatter gate has been defined to exclude very low and very high scatter signals. All the other plots on the layout are gated using this Scatter gate. Seven different volumes of CD64 were added to peripheral blood mononuclear cells and are shown in the seven color dot plots. To define the negative population for this titration experiment, a gate has been drawn on the lymphocyte population, shown in blue as the Lymphs gate, in the 2D plot of Side Scatter (SS Lin) versus CD64 (FL 8 Log). The positive population is the monocyte population which is defined in purple as the Monos gate. In the lower left corner of the layout is the Gate View window showing that the Lymphs and Mono gates are children of the Scatter gate, meaning the data is first gated using the Scatter gate and then the Lymphs and Mono gates are applied.

We will now define the custom tokens for each of the seven volumes of CD64 used in order to calculate a CD64 signal to noise ratio. The custom tokens will be defined as the ratio of the median CD64 fluorescent intensity of the monocyte population (the positive population) divided by the median CD64 fluorescent intensity of the lymphocyte population (the negative population).

 

 

The Custom Tokens window now appears on the layout. We will now dock the window to the left side of the layout. For more information on docking, please refer to the Docking tutorial.

 

 

The Custom Tokens window is now docked on the left side of the layout. We will now begin defining the custom tokens. For more information on creating custom tokens, please refer to the Using Text Boxes and Tokens tutorial.

 

 

A new Custom Token appears in the top half of the Custom Tokens window, shown highlighted in blue in Figure T17.5.

 

 

 

 

 

 

 

The Insert a Token dialog appears (Figure T17.8).

 

 

The Create Statistic dialog, with the Data Source category, appears, similar to Figure T17.9. This is where we will select the data source to use in the custom token definition.

 

 

 

The Statistic page of the Create Statistic dialog now appears, similar to Figure T17.10.

 

 

The Custom Tokens definition of '0.156 ratio' updates to reflect the changes made, shown in Figure T17.11.

 

 

 

 

We have now defined the numerator, the positive population, of the 0.156 ratio custom token. Now we will define the denominator, the negative population, of the custom token.

 

Alternatively the text can be copied using the pop-up menu.

 

 

Alternatively the text can be pasted using the pop-up menu.

 

The Custom Token formula definition updates to reflect the copying and pasting of the numerator text statistic to the denominator. We will now edit the denominator definition.

The Edit Statistic dialog appears, similar to the Create Statistic dialog previously shown in Figure T17.9. We will now modify the definition to use the Lymphs gate; we do not need to edit the Data Source page because we are still using the same plot.

The Statistic page of the Edit Statistic dialog appears, again similar to the Create Statistic dialog previously shown in Figure T17.10; however, this time we will select the Lymphs gate.

The Custom Token formula for 0.156 ratio updates to reflect the changes made, as shown in Figure T17.12.

 

 

We will now create a Custom Token formula for the 0.313μl CD64 data. However, instead of using copy and paste to define the Custom Token, we will drag and drop the color dot plot of 0.313μl CD64 onto the lower half of the Custom Tokens window.

 

 

The Paste Special dialog will appear as shown in Figure T17.14.

 

 

The Statistic page of the Edit Statistic dialog appears, again similar to the Create Statistic dialog previously shown in Figure T17.10. Again,n we will use the same statistic settings shown in Figure T17.10 to define the numerator of 0.313 ratio Custom Token. The Data source has already been set to the 0.313μl CD64 plot when we dragged and dropped the plot onto the Custom Tokens definition window. Refer to Figure T17.10 for the following steps

We have defined the numerated for the 0.313 ratio Custom Token. Now, we will define the denominator, again dragging and dropping the plot of 0.313ul CD64.

The Paste Special dialog will appear again, as previously shown in Figure T17.14.

The Statistic page of the Edit Statistic dialog appears, similar to the Create Statistic dialog previously shown in Figure T17.10. Again, we will use the Y-median as the statistics option. However, now we will select the Lymphs gate to define the denominator.

The Custom Tokens window should now appear similar to Figure T17.15, with the definitions for Custom Tokens for 0.156 ratio and 0.313 ratio.

 

 

Now we will define the Custom Token for the 0.625μl CD64 data. Instead of defining the Custom Token from the beginning, we will copy and paste the definition of the 0.313 ratio Custom Token.

The 0.625 ratio Custom Token has now been defined to use the 0.625μl CD64 data. We will now use copy and paste in the pop-up menu to define the 1.25 ratio token using the definition of the 0.625 ratio Custom Token.

 

 

 

 

The 0.625 ratio custom token now appears twice. We now need to edit both the name of this custom token and the definition to use the 1.25μl CD64 plot data.

 

 

Continue to define the Custom Token ratios for the 2.5μl, 5μl, and 10μl CD64 plots using the previous copy and paste instructions. Repeat instructions 75 through 92 substituting 2.5μl for the 1.25μl data, and then the 5μl and 10μl data, until all the Custom Tokens have been defined. The final Custom Token window should look similar to Figure T17.19.

 

 

 

 

In the next section, we will create a custom data table.