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:
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.
Celigo produced the following results for MDA-MB-231 and K562: percent dead cells after 24 hours of Benzethonium drug treatment
Graphing
- 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.
- The results are shown below:
- 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