Proliferation

Using flow cytometry to track cell generations is now possible, thanks to the introduction of cell tracking dyes. FlowJo presents a graphical display and a table of data on each generation in the subset. This will provide you with information about how many cell divisions have occurred. In addition the FlowJo Proliferation Platform draws gates in order to separate each generation.

Start by clicking on the population to analyze and selecting Proliferation from the Platform menu.

This brings up the Proliferation Platform.

Select a parameter on the X-axis.

The default number of peaks is 8, although it is important that the chosen number of peaks exceed the actual number of peaks in the data.

It is useful to initially calculate the model without constraints (adding constraints as necessary to obtain the best fit).

1) Calculate the model by clicking on the Calculate button.

Click a yellow arrow to cycle through samples.

Click the black triangle to select a sample.

Click the clipboard icon to copy table to clipboard.

The Division Index*, Proliferation Index*, and the %Divided* are explained below.

The better the model fit, the lower the Root Mean Square (RMS).

2) Create Gates...Gates dividing the generations can be created by clicking the Create Gates button. (Draws a gate halfway between each peak and adds them to the Workspace.)

3) Show Overlay: Select this button to open the Multigraph Overlay window showing each generation's gate displayed on a bivariate plot with the parameters of your choice.

*Division Index is the average number of divisions that a cell (that was present in the starting population) has undergone. For example, if half of the cells in the starting population divided and the average number of divisions was 4, the Division Index would be 2.

*Proliferation Index is the average number of divisions that those cells which divided underwent. For example, if the average number of divisions for all the cells was 4, the Division Index would be 4.

*%Divided is the percentage of the cells of the original sample which divided (assuming that no cells died during the culture). For example, if half of the cells in the starting population have divided, the %Divided = 50%.

These statistics are related in the following way: Division Index = (Prolif. Index)(%Divided)

The Model Parameter Adjustments can be used to obtain a better fitting model.

As with all other platforms in FlowJo, the Proliferation Node can be applied to groups of samples by dragging.

Each generation can be analyzed separately (double click the generation number to open a graph).

Select the Backgate icon to display a backgating analysis of the generation.