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
Goal:
Detect and quantify dead cells using PI stain in adherent MDA-MB-231 and suspension K562 cell lines
Protocol:
- 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
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 for 24, 48 and 72 hours
Typical images and fluorescent object identification looked as shown below for PI-stained cells “Graphic Overlay” segmentation
Results for the K562 and MDA-MB-31 counts of dead cells after 24 hours of Benzethonium drug treatment
Graphs
- 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
- IC50 values were calculated with Graph Pad Prism
- Cell death increased over time with Benzethonium (14.5 µM) versus the control
Conclusion
- 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