Use of 5-Ethynyl-2′-Deoxyuridine Labelling and Flow Cytometry to Study Cell Cycle-Dependent Regulation of Human Cytomegalovirus Gene Expression
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The cell cycle position at the time of infection has a profound influence on human cytomegalovirus (HCMV) gene expression and therefore needs consideration in the design and control of HCMV experiments. While G0/G1 cells support the immediate onset of viral transcription, cells progressing through the S and G2 cell cycle phases prevent HCMV from entering the lytic replication cycle. Here, we provide two fast and reliable protocols that allow one to determine the cell cycle distribution of the designated host cells and monitor viral protein expression as a function of the cell cycle state. Both protocols make use of the thymidine analogue 5-ethynyl-2′-deoxyuridine and “click” chemistry to label HCMV-non-permissive S phase cells in a gentle and sensitive way.
Key wordsCell cycle Cytomegalovirus EdU labelling Flow cytometry HCMV antibodies Immediate early genes Propidium iodide staining
This work has been supported by the DFG grant WI2043/3-1 to L.W. and C.H.
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