Purpose Perform kinetic viability assay on MDA-MB-231 and K562 cells
Existing Method(s) Cell Titer Glo, Flow Cytometry
Target Cell Type MDA-MB-231 adherent and K562 suspension cells
Experiment Plan Scan plate using Red and Bright field channels
Hypothesis Determine the counts of PI-positive cells kinetically at 24, 48 and 72 hours

Celigo Setup

Plate Type Greiner 781091 384-well black wall clear bottom
Scan Channels Red, Bright field
Resolution 1 µm/pixel
Scan Area Whole well
Analysis Method Target 1 + 2
Scan Frequency Daily, for 3 days
Scan Time ~15 minutes

Assay Protocol and Plate Setup


Detect and quantify dead cells using PI stain in adherent MDA-MB-231 and suspension K562 cell lines


  • Seeded MDA-MB-231 at 2,000 cells/well and allowed to incubate overnight
  • Suspension cells were plated the day of experiment with a working solution of 2X PI at 3,000 cells/well
  • Prepared Benzethonium at 25 µM final concentration and serially diluted by 1.3 dilution factor
  • Removed media and added drug, control and PI stain to wells
  • Incubated the plate for 24, 48 and 72 hours with drug and dye
  • Imaged the plate using the Celigo image cytometer

Plate map for Benzethonium (µM) drug treatment and PI staining

plate setup drug treatment


Drug-treated MDA-MB-231 and K562 cells showed an increase in PI positive cells

  • PI-positive cells were determined by staining the cells for 24, 48 and 72 hours

Typical images and fluorescent object identification looked as shown below for PI-stained cells “Graphic Overlay” segmentation

cell images of drug treated cells

Results for the K562 and MDA-MB-31 counts of dead cells after 24 hours of Benzethonium drug treatment

well level data adherent suspension


  1. Generated a graph using Microsoft Excel comparing 25 µM Benzethonium to the control after 24, 48 and 72 hours of treatment. In this example, the average of 4 data points were plotted

kinetic viability plots

  • IC50 values were calculated with Graph Pad Prism

cell death plots

  • Cell death increased over time with Benzethonium (14.5 µM) versus the control


  • The Celigo successfully performed PI viability assay using MDA-MB-231 and K562 cell lines drug treated with Benzethonium
  • Performed kinetic viability assay using PI allowed for the enumeration of total number of PI-positive cells over a period of 24, 48 and 72 hours