The aim of this investigation is to identify the temporal activation of intracellular signaling proteins in response to TLR activation. Studies are performed in murine BMDM, as the recent identification of mutant mice deficient in TLR responsiveness provides an excellent tool to dissect the intracellular signaling cascade.
Flow cytometry techniques are being used characterize the cell signaling pathways, identified by the Genomics, Computational, and Forward Genetics cores, that serve to activate and propagate the transcriptional networks in response to priority pathogens.
Flow Cytometry
Flow Cytometry techniques are used to study multiple parameters that
can be
interrogated through measurement of light including light scatter characteristics and fluorescence measurements.
Flow cytometry allows the analysis of multiple parameters on a single cell level and allows the characterization of
populations of cells based each cells individual characteristics. The Nolan laboratory has developed a technique
known as fluorescent cell barcoding (FCB) that allows multiple populations of cells to be interrogated
simultaneously. In this technique, different populations of cells (for example, cells from different points in a
time course) are stained with various concentrations of fluorescent dyes so that the different populations can be
combined and then stained and analyzed for specific parameters. For example, fluorescent antibodies that are
specific for various phosphorylated and unphosphorylated signaling molecules can be using to analyze signaling
cascades.
Flow Cytometry and the Innate Immune Response
We are using flow cytometry techniques
to study
the regulation of the innate immune response to whole pathogens and pathogen-associated molecular pattern
(PAMPs) that are known to activate Toll-like receptors. We are focusing on protein phosphorylation because this is a
major mechanism by which immune receptors activate transcriptional responses. We are performing a comprehensive
kinetic analysis of early phospho-signaling events in macrophages and dendritic cells stimulated in vitro
with
priority pathogens and their PAMPS. We are also analyzing phospho-protein levels in immune cells isolated from
wildtype and ENU-mutagenized mice generated by the Forward Genetics cores challenged in vivo with priority
pathogens
and their PAMPs. Raw data provided under the "Experimental Signaling Data" link can be processed and displayed using
the flow cytometry analysis software available through the CytoBank
database at Stanford University (Nolan Lab, Cell Signaling Core).