S-phase analysis using bromodeoxyuridine uptake
The determination of the percentage of cells in S-phase is dependent upon the detection of a thymidine analogue, bromodeoxyuridine (BrdU), which when added to culture medium is incorporated into DNA during DNA replication and bivariate analysis of total DNA content using propidium iodide staining along the X-axis plotted against BrdU incorporation detected via FITC labelled antibodies to BrdU on the Y-axis. Immediately following a pulse of BrdU labelling, the green fluorescence (BrdU content) is observed in S-phase. If the population is sampled every 1 h to 2 h, the passage of labelled cells into G2 can be observed followed by the appearance of green fluorescence in G1 as the cells pass through mitosis. The fluorescence intensity is halved as the cells divide because the average amount of BrdU per cell is halved. Green fluorescence reappearing in S-phase is indicative of a complete cell cycle and the time taken is indicative of the cell cycle kinetics for the cell population sampled. Depending on the cell type, the BrdU pulse can be as short as 6 min and still be detectable by flow cytometry.
- Cell cycle analysis of Chinese hamster ovary (CHO) cells
- BrdU detection using DNase
- BrdU detection by antibody Protocol
- DNase Protocol
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