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.

There are two data files in this workspace. Both represent sorted CD4 Memory T Cell populations. Both were stained with CFSE, an intracellular dye used to measure cell proliferation, and then cultured for three days. The first sample is an unstimulated control. The second has been stimulated with beads coated with anti-CD3 and anti-CD28. The unstimulated cells have not divided and all have a similar amount of CFSE fluorescence. 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. We have created a Lymphocyte population, and a second gated population to remove the beads.

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

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).

Calculate the model by clicking on the Calculate button.

Click an arrow to cycle through those samples 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 Division Index, Proliferation Index, and the %Divided are explained below.

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).