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
Goal:
Detect and quantify dead cells using PI and Hoechst stains in adherent MDA-MB-231 and suspension K562 cell lines
Protocol:
- 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:
Results
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.
Celigo produced the following results for the MDA-MB-231 and K562 counts of percent dead cells after 24 hours of Benzethonium drug treatment
Graphing
- 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.
Conclusion
- 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