Phospho-specific flow cytometry (phospho-flow)

Getting started with phospho-flow?  Try taking “the U937 challenge“.  This is a great starting phospho-flow experiment because it is simple (stimulate a suspension cell line with a few different cytokines, measure a few abundant phospho-proteins) and we’ve done it over and over, so we know what to expect.  We teach this experiment in courses and it’s the first experiment that all Irish lab rotation students do.

The following is a manual from a 2008 course on phospho-flow.  It is designed around groups of students picking from one of three types of experiments: (1) titration of a cytokine, (2) a signaling timecourse, and (3) a mini-profile of cytokine stimuli vs. phospho-protein readouts.

Full Manual – PhosphoFlow Course – Irish

Note: The volumes listed in this manual are for a 1 mL stimulation volume, which is OK in a classroom setting, but too large for day to day use on primary samples.  For primary cells, we recommend scaling the experiment to work for 2 million primary cells in 200 uL (10 x 10^6 cells / mL).  The protocol from our PNAS 2010 manuscript about abnormal B cell receptor signaling networks in Follicular Lymphoma has an updated version of the manual.

Irish et al., PNAS 2010 manuscript with detailed phospho-flow and fluorescent cell barcoding methods in Supporting Information (p9 and p10).  I’ve also pulled out a list of clones and sources for phospho-specific antibodies used in this work.

Once you’ve done this protocol, you may wonder:

• How do I compare stimulated and basal levels of signaling?
• What is the correct scale transformation for my data?

Other Cytometry & Phospho-flow Resources

• Cytobank flow and phospho-flow analysis support & help pages
• BD Biosciences fluorescence spectrum viewer
• Invitrogen spectrum viewer
• BD FACSelect buffer compatibility resource
• Invitrogen labeling kits
• Purdue cytometry discussion

Questions?

For protocol questions or PDF reprint requests, e-mail Dr. Jonathan Irish