HCS image showing live, dead, and total cells in different colors

Optimized, automated assays for HCS

Membrane integrity is a commonly measured parameter in cell viability assays. Cells with compromised membranes allow entry to otherwise cell-impermeant DNA-binding dyes, and this DNA staining serves as a cell death indicator that can be used as the basis for a single-channel assay. Invitrogen Image-iT DEAD Green Viability Stain (green; Cat. No. I10291) is an ideal dead cell stain because it will label the nuclei of dead cells and also survives fixation and permeabilization so that it can be readily multiplexed with other techniques. In the example below, Image-iT DEAD Green is multiplexed with Invitrogen Hoechst 33342 (blue; Cat. No. H3570)  to give a total cell count and Invitrogen Click-iT Plus EdU to measure cell proliferation.

Typical viability assay

Live-cell assay protocol. Two-channel assays can be multiplexed with numerous additional parameters.

Imaging mode

  • Widefield
  • 10x magnification

Automatically measured properties



  • Total cells
  • Dead cells


  • Total cells
  • Live cells
  • Dead cells

Viability assay images

Eight-panel HCS image showing 3 viability parameters at 2 camptothecin concentrations

Single-channel viability assay with multiplexing. HeLa cells treated with camptothecin and stained with Invitrogen Hoechst 33342 (blue channel) for a total cell count. Dead cells are stained with Invitrogen Image-iT DEAD Green (green channel) and proliferating cells are labeled using Invitrogen Click-iT PLUS EdU with Invitrogen Alexa Fluor 647 picolyl azide (deep red channel).

Two-channel viability assay

Two-channel viability assay. A549 cells were treated with varying concentrations of camptothecin and labeled with the Invitrogen LIVE/DEAD Cell Imaging Kit. Living cells sequester the LIVE Green probe, whereas dead cells become permeable to the DEAD Red stain. Cells were counterstained with Invitrogen Hoechst 33342 for total cell count.

Sample experimental data

graph of dead cells vs camptothecin

Single-channel viability assay with multiplexing. Dose-response curve for HeLa cells treated with camptothecin, stained with Invitrogen Image-iT DEAD Green and measured using the Thermo Scientific CellInsight CX5 High Content Screening Platform.

graph of two viability parameters

Two-channel viability assay. Dose-response plot showing the effect of camptothecin concentration on cell viability using the Invitrogen LIVE/DEAD Cell Imaging Kit.