HIGH-CONTENT IMAGING
Cell cycle and proliferation
Where does cell-cycle progression change?
Combining DNA content with DNA synthesis and mitotic markers reveals more than a change in cell number. It separates cell-cycle populations and helps characterise the response to an anti-proliferative treatment.
EXPERIMENTAL DATA
The method
Label DNA synthesis
Allow EdU incorporation during a defined labelling period to identify cells synthesising DNA. Detect the incorporated label at the assay endpoint.
Measure complementary markers
Measure DNA content with a nuclear dye and use phospho-histone H3 to identify mitotic cells. A dead-cell stain can provide additional context.
Resolve populations
Use single-cell measurements to estimate G1, S, G2 and M fractions and compare their distributions across treatment conditions.
Reading the results
Single-cell scatter plots, cell-cycle phase fractions and mitotic index. DNA content alone resolves G1, S and G2/M; EdU and a mitotic marker add information on DNA synthesis and mitosis.
Interpretation
A phase distribution is an endpoint snapshot, not a direct measurement of cell-cycle speed. Treatment duration, labelling window and cell loss influence the observed fractions and should be considered together.
Study design
The cell model, exposure time, EdU labelling period and marker panel are matched to the research question. The illustrated 72-hour MCF7 experiment is one study configuration.