December 14, 2010  |  Cytobank, Education, Flow Cytometry  |  By  |  3 Comments

Biochemistry at the Single Cell Level

Dataset #811: 090902-PBMC stim with IL6, IL10, LPS (Public Version)

Western blots are great. That is, unless you’re studying signal transduction in a mixed population of primary cells. The amount of material necessary for many biochemical techniques is usually not practical when working with patient samples or rare cell populations.

Flow cytometry to the rescue! Phospho-flow tracks biochemistry at the single cell level. Simultaneous assessment of cell surface markers and signaling events for individual cells enable biologists to profile disease samples or monitor the effects of drugs in clinical samples.

See for yourself by trying out the PBMC Phospho-Flow Protocol. With the staining panels in this protocol, you will measure both intracellular signaling events and cell lineage markers. This combination enables you to track signaling in immune cell subsets – monocytes, B cells, CD4+ T cells, and CD4 T cells.

Download our protocol sheet and upload and analyze your data on Cytobank. You can also view sample data from this experiment on Cytobank. To interact with the sample data, you will need to make a clone of the FCS files or of the experiment. Watch our tutorial video for a step-by-step walkthrough on how to work with this data in Cytobank.

A brief note on compensation: compensation is a method to correct flow data for spectral overlap between detected fluorophores, such as antibody labels. As you might expect, compensation is a large topic unto itself, and we will discuss it in another blog post. What you need to know to work with dataset #811 is that a compensation matrix will need to be imported from the public experiment. The video tutorial will show you the necessary steps.

The results show clear differences in the activation of signaling pathways among immune cell subsets. For example, the heatmap plot of the data indicates that STAT3 was phosphorylated in all IL-6 stimulated cell types except B cells. Looking at view-through plots (highlighted in our recent post) or changing the plot type to histogram overlay plots reveal that the CD4 T cell population was heterogeneous in its response to IL-6. STAT3 was phosphorylated in about half of these cells.

Why are the colors different between the video tutorial and protocol sheet? It has to do with how the data is being displayed on the plots. You can change these settings using the options in the Plot Controls frame, located on the left side of the Working Illustration in a Cytobank Experiment. Change Equation to “Raw” to display calculated raw values of medians (as in the video tutorial); change Equation to “Log10 Ratio” to display fold change in phosphorylation relative to unstimulated controls (as in the protocol sheet).

Haven’t tried phospho-flow before? We recommend getting started with the U937 experiment.

– Stephanie