2D Cell Cycle (BrDu vs PI)

This model enhances the resolution of the events of the S phase from those in G0/G1 and G2/M and allows for the fitting of any prominent population, be it sub-G1, multiploid or early or late S phase.

If you have labeled cells with a Tymidine analog such as BrdU, or another molecule that binds preferentially to S-phase cells, the 2D model will allow you to use this parameter simultaneously with DNA content to produce a more accurate fit.

About the Model:

The model utilized to fit the data is a mixture model, which uses multiple populations (each existing anywhere on a 2D plane with any covariance). Each population is not predefined to "look for" any specific dividing cell population.

Based on the median intensities of the data, an iterative process adjusts the model toward maximizing the log likelihood of the model fitting the data. When the model reaches a stopping criteria, component membership is assigned based on "most likely population membership".

The model then is able to display the components as a single population, joining and naming the components using a "closeness" measure heuristic. This heuristic is based on what is known about the biology, but scaled to the individual sample.

Using 2D Cell Cycle:

To display data in the 2D platform, right click on any sample in the workspace and choose Add 2D Cell Cycle... The platform window shown upper right will appear.

FlowJo should identify both the BrDU and PI parameters and display them. If FlowJo does not identify them, select them from the parameter drop down menus.

Once your data is displayed, check Show model components (under the Statistics disclosure triangle) to display the clusters that the platform has found and adjust the number of components to what is expected based on the biology of your sample.

You can change the color of your components by clicking on the colored box of the component and selecting from the color palette.

Once you have identified your populations, you can choose to create gates based on those populations by clicking on the Create Gates button. Gates will appear in the workspace and can be utilized like all population nodes in FlowJo.

Use Boolean Gates to combine created gates to make an S Phase gate. Using 8 components for example, isolates debris into separate clusters, but also divides the S Phase. Combine gates 1, 3, 4 and 5 with a Boolean OR gate. This gives cleaner G1, S and G2 clusters.