Endpoint viability using DRAQ7 and Hoechst

DOWNLOAD
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 Far Red, Bright field and Blue channels
Hypothesis Drug treatment will increase the percentage of DRAQ7-positive cells

Celigo Setup

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

Assay Protocol and Plate Setup

Goal:

Detect and quantify the dead cells using DRAQ7 and Hoechst total stain in adherent MDA-MB-231 and suspension K562 cell lines

Protocol:

  • Seeded MDA-MB-231 and allowed to incubate overnight. Suspension K562 cells were plated on the first 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 according to the plate map
  • Incubated the plate for 24, 48 and 72 hours
  • Prepared a dye mix solution of DRAQ7 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:

endpoint viability plate set up

Results

Drug-treated MDA-MB-231 and K562 cells showed an increase in DRAQ7-positive cells

  • DRAQ7-positive cells (dead cells) were determined at the endpoint of 24, 48 and 72 hours by DRAQ7 and Hoechst as a total nucleated stain

Gate Plots for DRAQ7 Positive Cells:

Example of gating settings for Hoechst and DRAQ7-stained MDA-MB-231 adherent cell line, with blue graphic overlay outlining all objects and red graphic overlay outlining DRAQ7 cells. A similar setup was followed to work with suspension cells.

endpoint viability gating

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

endpoint viability plate level data

Graphing

  1. Graphs were generated using Graph Pad Prism for the dose-response of Benzethonium after 24, 48 and 72 hours of treatment. In this experiment, the average of 4 data points were plotted.
  2. The results are shown below:

endpoint viability dose response

  • IC50 values were calculated using Graph Pad Prism

Conclusion

  • Drug treated MDA-MB-231 and K562 cell lines were successfully imaged and analyzed on Celigo
  • Endpoint viability assay using DRAQ7 and Hoechst allowed for the enumeration of total counts and percentages of DRAQ7-positive cells
  • Acquisition of high-resolution DRAQ7 and bright field images of 384-well plate took about 15 minutes
2018-10-11T19:11:07+00:00