Overview of FCB Technique
As outlined in the figure below, sample one was unstimulated, sample two was stimulated, and sample three was treated with a small-molecule inhibitor before stimulation. After fixation, cells in standard phospho flow (left) were permeabilized with cold methanol, washed and stained with phospho-specific antibodies. In the FCB technique (right side), each sample was permeabilized with 20–25 °C methanol containing a different concentration of amine-reactive fluorescent dyes (FCB markers), yielding a unique fluorescence signature for each sample. Samples were then washed, combined into one tube, and stained with antibodies. During software analysis of the acquired data, the samples were deconvoluted back to the original samples based on their FCB signature. In both standard and FCB phospho flow techniques, fluorescence of the phospho-specific antibody in each sample was measured. In the plots, dotted lines indicate autofluorescence and red histograms represent sample fluorescence.
REFERENCES
1. 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.
