Flow Cytometry of Phospho-Proteins in Immune Cells
Newsworthy Events
Nolan GP.
2008. Featured Scientist Interview by ScienceWatch.com. Tracking Trends and Performance in Basic Research. April 2008
Lee AW, Sharp ER, O'Mahony A, Rosenberg MG, Israelski DM, Nolan GP , Nixon DF. Single cell, phosphoepitope-specific analysis demonstrates cell type- and pathway-specific dysregulation of Jak/STAT and MAPK signaling associated with in vivo HIV-1 infection. J Virol. 2008 Jan 23; [Epub ahead of print].
Krutzik PO, Crane JM, Clutter MR, Nolan GP. High-content single-cell drug screening with phosphospecific flow cytometry. Nat Chem Biol. 2008 Feb;4(2):132-42. Epub 2007 Dec 23.
Shachaf CM, Perez OD, Youssef S, Fan AC, Elchuri S, Goldstein MJ, Shirer AE, Sharpe O, Chen J, Mitchell DJ, Chang M, Nolan GP, Steinman L and Felsher DW. Inhibition of HMGcoA reductase by atorvastatin prevents and reverses MYC-induced lymphomagenesis. . Blood. 2007 Oct 1;110(7):2674-84. Epub 2007 Jul 10.
We have created systems that allow for measurement of phosphorylation states within cells by Flow Cytometry. The techniques can be applied to primary cells in conjunction with surface markers to delineate subsets. As such, we can confidently and rapidly undertake biophysical measures of signaling status within cells.
This section of the Proteomics Web site incorporates protocols for undertaking phosphoprotein analysis by Flow Cytometry, considerations for experimental design using phospho-flow, and sample data we have generated. We are soon to add a web board for discussing techniques and problems with others in the community and a database of phospho antibodies and surface markers we commonly use.
Rationale:
There is a need to measure complex populations of immune system cells and phenotype them not only for their cell lineage status but also for their relative activation state. Moreover, there is a need to link such data to phenotypic outcomes and to other data sets gathered on different instrumentations.
Changes in intracellular protein levels, subcellular localizations, or activation states are considered to be reflective of a cell’s capabilities or functions. Some of these events are transitory—such as some phosphorylation sites on target proteins. Some of the relevant cell populations are so rare as to make their isolation for standard biochemical analysis nearly impossible.
This project will first create and validate a series of probes for important phosphoproteins. There will be an initial emphasis on kinases whose regulation is dependent on phosphorylation (either self-phosphorylation or that imposed by an outside kinase). There will be a later inclusion of phosphoproteins whose activities are considered relevant to immune lineage function. The techniques we will use employ our recently developed staining procedures applying monoclonal and polyclonal antibodies against phosphorylation sites. The antibodies themselves are obtained commercially from a variety of sources and we have already verified the specificity of several in our published studies (Perez and Nolan, Perez et al Immunity).
Our goals for the Proteomics Center include:
- To create and verify up to 40 phosphoprotein probes. We will define sets of anti-phosphoprotein antibodies that can be used in conjunction with surface markers for analysis in immune cell lineages.
- To use reagents defined in Specific Aim 1 to analyze phosphoprotein levels in autoimmune lineage subpopulations. Sample analyses of these will be placed on the web along with downloadable datafiles and analysis programs.
