Users of the Proliferation and Cell Cycle Platforms have undoubtedly noticed that the statistics the model produces do not transfer exactly to the workspace when Gates are created from the model.

For example, the figure below shows a Watson-Pragmatic cell cycle model for one of our demo files. The associated statistics are noted on the model. The stats pertinent to this discussion are the G_{0}/G_{1}, S, G_{2}/M frequencies which in the model are roughly 57% / 24% / 18%.

If you were to press the **Create Gates** button at the top of the model window, and accept the “Stage” prefix you would get the workspace shown below the figure. Notice that the frequencies are roughly 64% / 14% / 21%. Why have 7% of the cells “shifted” from the S phase into the G_{0}/G_{1} and another 3% shifted from S into the G_{2}/M?

The statistics are actually calculated in two different ways. When using the models the frequencies are calculated using probabilities of membership, based on the model fit. For cells with intensity measured within a given range, the model can be used to determine the prob

ability of the cell belonging to each of the identified populations plus sub-G_{0}/G_{1} and super G_{2}/M. The probabilities of all cells are then added up to computethe phase probabilities for the entire sample. The summation of these probabilities will be another probability. To create fractions that add up to ~100% a normalization step is required. To preserve the relative relationship between phases we do not simply normalize to 100% but rather map the probabilities to those that sum to 100%. This process can sometimes lead to small negative numbers, particularly in the sub-G0/G1 or super G2/M populations. In these cases it is reasonable to think of a population with a negative frequency as simply a very small number of cells.

When you press the **Create Gates** button FlowJo places hard line gates on the model at the points where it becomes less probable that a cell belongs to one population and more probable that it belongs to another. Cells that are in the gate are treated as having a probability of membership of 100% for the “winning” phase and 0% for all other.

You’ll notice in the above example that the difference in the statistics is that the gated frequencies do not take into account that there are probably a decent number of S phase cells that spill into the other two distributions, so if you have the opportunity to use the model statistics, do so. The stochastic approach models the data better. However, if you use the model created subpopulations, continue to add layers of gates, and in the end need to come up with fractions that add up to 100%, you will need to use the gate frequencies.

- Bad Mo'Flow

Send comments and questions to john@treestar.com

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