November 17, 2010  |  Cytobank, Flow Cytometry  |  By  |  1 Comment

Take the U937 Challenge!

Dataset #61: Welcome to Cytobank – U937 dataset

So you want to do phospho-flow? Take the U937 challenge!

What is phospho-flow? It’s a specialized form of flow cytometry that enables biologists to probe signaling activity inside the cell. Flow cytometry has been widely used to analyze cell populations by probing the proteins that are outside on the cell surface. Phospho-flow cytometry includes a few extra steps – stimulation, fixation, and permeabilization – that enable researchers to also probe the transient phosphorylation state of intracellular signaling proteins.

The U937 experiment is the go-to training experiment for phospho-flow cytometry and has been performed hundreds of times by researchers around the world. What makes U937 cells so great? They respond robustly and consistently to cytokine stimulation and are thus an easy model system on which to try your first phospho-flow experiment.

Download our U937 Phospho-Flow Protocol sheet to get started. We encourage you to upload, analyze, and share your data with us on Cytobank. Sample data from the U937 experiment are also available on Cytobank – search for the public experiment titled “Welcome to Cytobank – U937 dataset”. To interact with the data, you will need to clone the experiment. For more information, watch the U937 tutorial video below or consult the U937 tutorial on our documentation site.

Note that “Welcome to Cytobank – U937 dataset” contains a few more experimental variables than the experiment outlined on the protocol sheet. To access only the relevant data shown in both the protocol sheet and the tutorial video, look for the public experiment titled “U937 dataset (Protocol Sheet adaptation)”.

Using the histogram overlay plot type to view the data allows us to see the response to stimulus for each of the phospho-proteins when compared to the unstimulated or basal state. If you’ve stimulated, fixed, and permeabilized the U937 cells properly, you should see that p-Stat1 has a large response (i.e., a large increase in phosphorylation) to IFNγ and p-Stat6 has a large response to IL-4. You can also view this illustration on Cytobank.

Did you meet the U937 challenge?

– Stephanie