HCS image showing nuclear segmentation and histone H3 localizationOptimized, automated assays for HCS

Cell cycle assays and mitotic index measurements provide insight into cell division and quantify the effects of compounds or treatments that impact mitotic progression. Automated assays can measure cellular DNA content as an indicator of where in the cycle a cell was stained, or you can detect the levels of proteins associated with mitosis such as Histone H3. An automated combination of both measurements will generate a more stringent assay. Mitosis-associated proteins are generally detected using an immunofluorescence assay, and DNA content can be measured using DAPI or other DNA stains depending on your wavelength and multiplexing requirements. A two-channel assay will report on both components at a choice of wavelengths, keeping the remaining channels free for multiplexing other parameters.

Typical cell cycle assay

Fixed-cell immunofluorescent assay protocol.

Imaging mode

  • Widefield
  • 10x magnification

Automatically measured properties

  • DNA content
  • Intensity levels of up to 3 secondary targets
  • Correlation at single-cell and population level

Cell cycle assay images

Cell cycle assay. A549 cells were treated with increasing doses of a mitotic inhibitor for 24 hours. The cells were stained for phospho Histone H3 and detected using an Invitrogen Alexa Fluor 488 secondary antibody. Invitrogen HCS NuclearMask Deep Red stain was used as a nuclear segmentation tool.


Sample experimental data

Dose-response plot of mitotic inhibitors in A549 cells using a non-linear regression with GraphPad Prism software to determine an EC50.