how to use R to analyze cell cycle with propidium staining by flow cytometry

1. Import your data into R:

You can import your flow cytometry data into R using the flowCore package. First, install the package using the following command:

install.packages("flowCore")

Then, load the package and use the read.flowSet() function to import your data:

library(flowCore)
flow_data <- read.flowSet("path/to/your/data.fcs")

2. Preprocess your data:

Before analyzing your cell cycle data, you need to preprocess it to remove any noise or artifacts. You can do this using the preprocessFlow() function from the flowCore package. This function performs several preprocessing steps, including compensation, transformation, and gating:

preprocessed_data <- preprocessFlow(flow_data)

3. Gate your data:

Next, you need to gate your data to select the population of cells you want to analyze. In the case of cell cycle analysis, you typically want to select the singlet population of cells and exclude any debris or dead cells. You can use the flowWorkspace package to create gating strategies and apply them to your data:

install.packages("flowWorkspace")
library(flowWorkspace)
gs <- GatingSet(preprocessed_data)
plot(gs)

This will open a graphical interface where you can create and apply gating strategies to your data.

4. Calculate cell cycle phases:

Once you have gated your data, you can calculate the percentage of cells in each phase of the cell cycle using the cellcycle package. This package provides several methods for estimating cell cycle phases from propidium staining data:

install.packages("cellcycle")
library(cellcycle)
cycle_data <- cellcycle(gs, "Pr DNA1", "Pr DNA2")

Here, "Pr DNA1" and "Pr DNA2" refer to the channels in your flow cytometry data that contain the propidium staining signal.

5. Visualize your results:

Finally, you can visualize your results using plots or histograms. The ggcyto package provides tools for creating publication-quality plots from flow cytometry data:

install.packages("ggcyto")
library(ggcyto)
autoplot(gs, "Pr DNA1", "Pr DNA2") + geom_density_ridges()

This will create a density plot of your propidium staining data, with the different cell cycle phases highlighted in different colors