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.
Division 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).
Proliferation Index is the total number of divisions divided by the the number of cells that went into division. The proliferation index only takes into account the cells that underwent at least one division, that is, only responding cells are reflected in the proliferation index. This is probably a more useful value to compare from sample to sample, as it considers only the fraction of responding cells.
The proliferation index more faithfully reflects what the biology of the responding system is; the division index reflects what the entire system is doing.
Another way to think about it - between the two, whichever value is smaller is the average number of divisions of all cells (including nonresponders); the larger value is therefore the average number of divisions for the responding population.
%Divided is the same as the Precursor frequency.
Peak CV is the coefficient of variation for the peaks. The same value of CV is modeled for all peaks. Typically, this value is 4-7%.
Peak ratio is the ratio of fluorescence between subsequent peaks. i.e., 0.5 implies that peak "n" has half as much fluorescence as peak "n - 1". Values > 0.5 are not biologically meaningful, and usually arise when the log amp is not very good. The value should be close to 0.5.
As an example of how Proliferation Index and Division Index would be calculated, consider the following:
G0 = 15888 G1 = 32922 G2 = 13647 G3 = 897
The number of cells at start of culture: 15888 + (32922/2) + (13647/4) + (897/8) = 35872.875
The total number of divisions: (32922/2)*1 + (13647/4)*2 + (897/8)*3 = 23620.875
The number of cells that went into division: 35872.875 - 15888 = 19984.875
Division Index: 23620.875 / 35872.875 = 0.66
Proliferation Index: 23620.875 / 19984.875 = 1.18
If the model does not fit the data in a preferrable way, 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).
