The FlowJo Proliferation Tool presents a graphical display and a table of data on each generation in the stimulated sample. This will provide you with information about how many cell divisions have occurred. In addition the tool draws gates in order to separate each generation.
In this example, a sample is stimulated with beads coated with anti-CD3 and anti-CD28. With each cell division, the CFSE is divided between the two daughter cells. Therefore the amount of CFSE fluorescence shows the number of divisions any given cell has undergone. First a Lymphocyte population is gated, and a second gate is made to remove the beads.
Start by clicking on the population to analyze and selecting Proliferation from the Tools menu.This brings up the Proliferation Platform. Mouse over the red dots to learn more.
Select an available parameter for the X-axis from the drop-down menu.
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.
Calculate the model by clicking on the Calculate button. The tool will then appear like the next image, below. It is useful to initially calculate the model without constraints (adding constraints as necessary to obtain the best fit).
Click an arrow to cycle through other samples in the Workspace with proliferation applied. The green line is the Model Sum. To view it, check the Draw Model Sum check box.
Select Edit to copy the graph with or without the colored model fit information, or copy just the column of statistics. The better the model fit, the lower the Root Mean Square (RMS).Gates dividing the generations can be created by clicking the Create Gates button. (Draws a gate halfway between each peak). The gates are added to the Workspace.
Show Overlay: After you Create Gates, select the Show Overlay button to open the Multigraph Overlay in the Layout editor, showing each generation's gate displayed on a bivariate plot with the parameters of your choice. At right, a contour plot of each subpopulation on Forward vs. Side Scatter.
Division Index is the average number of cell divisions that the responding cells underwent. (i.e., ignores peak 0). This is probably a more useful value to compare from sample to sample, as it considers only the fraction of responding cells.
Example: if sample "A" has twice as many responding cells as sample "B", but the number of divisions that responding cells actually did was the same, then the proliferation index of sample "A" is twice as large as that for sample "B", but the division indices are the same.
The division index more faithfully reflects what the biology of the responding system is; the proliferation index reflects what the entire system is doing.
Proliferation Index is the average number of cell divisions that a cell in the original population has undergone. This is an average even for cells which never divided (i.e., includes the undivided peak).
%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%. "% Divided" is the same as the Precursor frequency.
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).
