FlowJo’s Proliferation tool looks for a pattern typically expressed by cells loaded with fluorescent dye and allowed to divide. A common probe for this assay is CFSE, but several others are available from vendors like Invitrogen.
FlowJo finds the original population and then models the division by looking for peaks with diminishing fluorescence with an approximate ratio of 0.5 per 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 Biology band, or else right-click and choose proliferation.
Show Overlay: After you Create Gates, select the Show Overlay button to open the Multigraph Overlay in the Layot 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 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 preferable 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).