It is expected that each time a cell divides each of the two cells in the next generation will have half as much dye, so their signal will be half as intense. Typically, FlowJo will set a peak ratio close to the expected 0.5 percent. The number of peaks also need to be adjusted to represent the number of peaks present in the analysis. These can be manually adjusted and fixed so that they are consistent across the proliferation analysis. When proliferation is first applied to a sample, FlowJo will try to estimate the optimal Peak Ratio and CV to represent the population. This node can be double clicked to open the proliferation window, or drag and dropped onto populations or groups to apply the proliferation analysis to other samples. When proliferation is first selected it will create a node in the workspace under the population proliferation is being preformed on. The “Generation 0” peak can be set by drawing a gate around the unstimulated peak in the proliferation assay. The generation 0 peak represents the undivided cells and should have a relatively consistent intensity and CV across the samples collected in the proliferation assay. Usually, proliferation assays will begin analysis using an unstimulated sample to determine the “Generation 0” peak. It is a good idea to do some initial clean up gating (size, live/dead, singlets, etc.) to isolate a population to preform proliferation analysis on. The Proliferation Platform can be found with other platforms under “Biology” in the “FlowJo” tab. A common probe for this assay is CFSE, but several others are also. FlowJo University has a great webinar that demonstrates how to use the proliferation tool. FlowJo’s Proliferation platform allows users to quantify many different common types of proliferation assays which look at cell division over time.įlowJo’s Proliferation tool looks for a pattern typically expressed by cells loaded with fluorescent dye and allowed to divide. Typically this tool is used to model the sample as it undergoes 3-6 divisions and generate related statistics.
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