HCS image showing proliferating and nonproliferating cells in different colorsOptimized, automated assays for HCS

Measuring the synthesis of new DNA is the most precise way to assay cell proliferation in individual cells, cell populations, or tissues. This automated assay measures new DNA synthesis based on incorporation of the nucleoside analog EdU into DNA. A copper-catalyzed “click” reaction adds a fluorescent Invitrogen Alexa Fluor dye conjugate to the EdU and enables detection with a range of wavelength choices for easy multiplexing. With a nuclear stain for the detection of all cells and an Alexa Fluor dye to label newly synthesized DNA, proliferation can be measured in a two-channel assay. All the remaining channels are available to measure other cellular parameters.

Typical cell proliferation assay

Fixed-cell assay protocol after EdU incorporation. Mild click reaction permits multiplexing with antibodies and common staining methods.

Imaging mode

  • Widefield
  • 10x or 20x magnification

Automatically measured properties

  • Total cells
  • Replicating cells

Cell proliferation assay images

A549 cell proliferation assay. A549 cells were treated with increasing doses of paclitaxel. Cells were stained with Invitrogen Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (red) and Invitrogen Hoechst 33342 stain (blue). Cells were imaged using the Thermo Scientific CellInsight CX7 High-Content Analysis Platform, and total cells and replicating cells were measured using Thermo Scientific HCS Studio Software.

Cell proliferation measurements can be multiplexed with GFP expression. U2OS cells expressing GFP are labeled with Invitrogen Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (red), and Invitrogen Hoechst 33342 (blue). Proliferating cells have red nuclei. Non-proliferating cells have blue nuclei.


Sample experimental data

Dose-response plots of U2OS cells treated with increasing concentrations of proliferation inhibitors and stained using Invitrogen Molecular Probes Click-iT Plus EdU labeled with Invitrogen Molecular Probes Alexa Fluor 647 dye.