Phosphoprotein Detection by High-Throughput Flow Cytometry
Phospho flow cytometry is a powerful technique for the detection of protein phosphorylation events that, like Western blotting, relies on phospho-epitope-specific antibodies. In contrast to the latter, however, multidimensional and directly quantifiable data is obtained at the single-cell level allowing separate analysis of small cell populations in complex cellular mixtures. Furthermore, up to 30 phospho-specific antibodies or antibodies identifying other posttranslational modifications in combination with cell surface markers can be analyzed in a single experiment. Utilizing a technique called fluorescent cell barcoding that enables combination of up to 64 samples into one tube for multiplex analysis and later data deconvolution, phospho flow cytometry is turned into a medium- to high-throughput technology.
Key wordsFlow cytometry Phospho flow Phospho-specific antibody Fluorescent cell barcoding Signaling
Our research is funded by grants from the Research Council of Norway, Norwegian Cancer Society, the European Commission (7th Framework Programme), South Eastern Norway Regional Health Authority, the K.G. Jebsen Foundation, and Novo Nordic Foundation which has permitted the method development as described here. Johannes Landskron is a Senior Scientist funded by the Jebsen Centre for Cancer Immunotherapy.
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