One of the most common methods used to study cell proliferation induced by different treatments is the dye dilution technique. In this technique, the cells of interest (usually T-cells) are labeled uniformly with tracking dyes such as CFSE or the CellTrace and PKH series of dyes. With each generation, the dye content of the cells decreases by half as the dye is equally divided among the daughter cells. Thus, as time progresses, cell populations that have divided can be identified by their characteristic distribution of peaks with diminished fluorescence as compared to the undivided control. The proliferation module in FCS Express performs sophisticated curve fitting in order to quantify the proliferation characteristics of your population. This allows for the easy determination of the proportion of cells that proliferated, as well as other derived statistics.

 

In this tutorial we will learn how to apply such algorithms, create proliferation statistics, format the plotted data, and generate publication quality reports in a batch process.

 

 

Layout Design and Gating Strategy Notes:

   

The layouts for use in this tutorial are listed below can be found in the Proliferation Tutorial folder within the Tutorial Sample Data folder.

 

•Proliferation Tutorial Fitting Proliferating Cells Section.fey

•Proliferation Tutorial Formatting Section.fey

•Proliferation Tutorial for Batch Processing.fey

 

The layouts have been optimized for ease of use while assuming that you are familiar with the basics of data analysis in FCS Express. The samples for the data set are listed below and were stained with SYTOX AADvanced (live/dead marker), CD4 Alexa 488, and CellTrace Violet (proliferation dye).

 

•Unstained Control.fcs

•10 uM CellTrace Violet Control.fcs

•10 uM CellTrace Violet Stimulated Control.fcs

•10 uM CellTrace Violet Stimulated Sample.fcs

 

    In the Gating page of the layouts, the top two plots define the live cells in the assay using a hierarchy of gates from the SSC-H, FSC-H, and SYTOX AADvanced-H parameters. The plots in the middle row of the Gating page are an inverse greyscale density plot (left) and a color dot plot (right). These plots are used to help visualize generational data and are useful for further downstream analysis of the proliferation data. The final plots for the analysis are two histograms of the CellTrace Violet-H parameter:  one which contains a marker that is linked to the Non Proliferating Control gate (M1) and is labeled Proliferation Histogram in the title;  the other, "Background Noise Histogram," contains a marker for Background Noise. (Note: The Background Noise histogram is only visible in the Proliferation Tutorial Fitting Proliferating Cell.fey while in the other layouts it can be found on a hidden page). The Proliferation Data page contains a pre-inserted proliferation plot of the data. The Proliferation Tutorial for Batch Processing.fey layout has completed all of the steps from the first three sections of the proliferation tutorial so you may proceed directly to batch processing the data.

 

We will begin by learning how to insert a proliferation plot and how to convert a histogram to a proliferation plot.