Purpose Perform endpoint viability assay on MDA-MB-231 and K562 cells treated with Benzethonium for 24, 48 and 72 hours
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, Bright field and Blue channels
Hypothesis Drug treatment will increase the percentage of PI-positive cells over time

Celigo Setup

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

Assay Protocol and Plate Setup


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


  • Seeded MDA-MB-231 and allowed to incubate overnight. K562 Suspension cells were plated on the day of the experiment
  • Prepared and serially diluted the drug Benzethonium to generate a dose response
  • Prepared the control with water in media
  • Added drug dose response and control to the wells
  • Incubated the plate for 24, 48 and 72 hours
  • Prepared a dye mix solution of PI and Hoechst in PBS
  • Added dye mix to the drug-treated wells and incubated the plate
  • Imaged the plate using Celigo image cytometer

Plate maps for Benzethonium (µM) drug treatment and Hoechst time point staining:

plate map 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 at the end point of 24, 48 and 72 hours with PI and Hoechst stains

Gate Plots for PI-Positive Cells:

Example of gating settings for Hoechst + PI stained MDA-MB-231 adherent cell line, with Blue graphic overlay outlining all objects and Red graphic overlay outlining PI-positive cells. Follow a similar setup for work with the suspension cells.

gate plots for pi positive cells

Celigo produced the following results for the MDA-MB-231 and K562 counts of percent dead cells after 24 hours of Benzethonium drug treatment

drug treatment plate level data


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

endpoint data graphs


  • The Celigo successfully performed PI viability assay using MDA-MB-231 and K562 cell lines
  • Performing an endpoint viability assay using PI and Hoechst allowed for the calculation of percentages from the enumeration of PI-positive and total cell counts
  • Acquisition of high-resolution PI and bright field images of a 384-well plate took ~ 15 minutes