Analysis of signaling pathways in innate immunity using fluorescent cell barcoding
Recent advances from the Nolan and other laboratories have enabled the development of a new generation of flow cytometer-based assays that improve measurement of phosphorylated proteins in signaling pathways. Fluorescent barcoding (FCB) combined with the intuitive grouping of data points along basic experimental parameters permit the creation of "FCB phospho-flow panel assays" that are rapid, versatile, and cost-effective.
Additional information about the FCB technique can be found here.
Experimental designs and topics that are well suited for FCB phospho-flow panel assays include:
- Isolated, fixed & permeablized cell types
- Experiments employing in vitro stiumulation: Time course or dose response assays
- Inhibitor studies (including drug development)
- Cascades of phosphorylated proteins in order to better define signaling networks, specifically in the fields of Immunology, Stem Cell Biology, Cancer Biology, Neurobiology and Others
A central strength of this assay is the coupling of informative panels of phospho-flow staining, FCB, and intuitive parameter groupings to facilitate the high throughput analysis of signaling pathways. This is achieved through the usage of “Primary and Secondary Groupings” that enable data points to be organized to query experimental questions with minimum time and resources. Primary groupings are the binary (or tertiary, etc.) segregation of experimental variables organized along intuitive hierarchies. For example: Mouse strain (see parameter I), agonist type (see parameter II), & phospho-protein staining target (see parameter III, below). Secondary groupings are data points that measure the effects of a series of a variable parameter, such as time or dose data points for a given stimulus, and are optimally suited for barcoding (see parameter IV, below).
Depending on the experimental questions of interest in a study, substitute targets or additional panels may be inserted. The selections below focus on TLR signaling in macrophages. Alternative staining panels can be assembled as needed for other cell types.
The time course points (TLR agonists- in vitro-BMDM) show above can be measured using a barcoding matrix of Pac Orange by Pac Blue, which permits the subsequent staining for phospho-flow targets with Ax647-A, Ax-488-A, and other conjugates.
Click here for more details on the experiment outlined above.
Analyzing the data involves several steps as outlined below. In this figure the end result, a histogram plot of time course data for phosphorylation of MAPKAPK2 in stimulated BMDM is achieved by:
a and b) Gating FSC (Forward) X SSC (Side)
c) Fluorescence barcoding with forward deconvolution to identify timepoint dimensions &
d) Generating a histogram plot of time course data for phosphorylation of MAPKAPK 2 in stimulated BMDM
Lastly the entire data set analyzing the phosphorylation of MAPKAPK2 (wt vs MyD88-KO, stimulated by TLR agonists: PAM 3, PolyIC, & LPS) is shown in a combined histogram/warm plot.
In summary, we describe a “FCB - panel assay”, wherein 396 separate conditions are consolidated into 66 stains. This conserves antibodies, time, and improves the precision of time course measurements. For the example shown, the data obtained with this Fluorescent Cell Barcoded Phospho-flow Panel Assay, regarding pMAPKAPK2 signaling downstream of TLR agonists, investigated in Wild Type vs. MyD88-KO mice, appears generally consistent with previously published studies, albeit with novel data concerning the kinetics of signaling.
REFERENCES
1. Krutzik P.O., Crane J.M., Clutter M.R., Nolan G.P. (2008). High-content single-cell drug screening with phosphospecific flow cytometry. Nat Chem Biol. Feb;4(2):132-42. Epub 2007 Dec 23.
2. Krutzik P.O. and Nolan G.P. (2006). Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling, Nature Methods 3, 361-368.
3. Edwards, B.S., Oprea, T., Prossnitz, E.R. & Sklar, L.A. Flow cytometry for high-throughput, high-content screening. Curr. Opin. Chem. Biol. 8, 392–398 (2004).
