Purpose | Monitor the effects of a panel of drugs on the apoptosis of U87MG Glioblastoma MCTS using Caspase 3/7 and Hoechst fluorescent staining |
Current Method(s) | Microscopy |
Target Cell Type | U87MG |
Experiment Plan | Allow U87MG spheroids to form and treat with a panel of drug compounds, then image on day 13 to measure the apoptotic effects of the compounds |
Hypothesis | Using the bright field and fluorescent imaging, the Celigo will rapidly provide multicellular tumor spheroid images, and measure Caspase 3/7 and Hoechst fluorescent intensities of treated U87MG MCTS |
Celigo Setup
Plate Type | Nexcelom U-bottom Ultra-low Attachment 384-well Plate (Cat# ULA-384U) |
Scan Channels | Green, Blue, and Bright Field |
Resolution | 1 µm/pixel |
Scan Area | Whole well |
Analysis Method | Tumorsphere 1 + 2 + Mask |
Scan Frequency | Endpoint |
Scan Time | ~8 min |
Assay Protocol and Plate Setup
Goal:
Image and analyze the apoptotic effects of a panel of drug compounds on U87MG MCTS on day 13.
Protocol:
Cell Preparation
- Seeded 500 U87MG cells/well in ULA 384-well plates
- On day 4, added different serially diluted drug compounds at 2x and a vehicle control in media
- On day 13, prepared and added Caspase 3/7 and Hoechst for staining the MCTS
- Incubated the plate at 37 °C and 5% CO2 for 60 min
- Imaged and analyzed on Celigo
- Compared the spheroid Caspase 3/7 fluorescent intensity for each drug compound at each time point to characterize the tested compounds
Plate map:
Data Collection
- After incubating the spheroids with drugs at different concentrations, the spheroids were stained with Caspase 3/7 and Hoechst
- The plate with stained spheroids was imaged on day 13
- The captured images were then analyzed in the Celigo software for the entire 384-well plate
Data Analysis
- The images were analyzed by using Tumorsphere 1 + 2 + Mask application to identify the MCTS in the well
- The fluorescent intensities of Caspase 3/7 were measured for each drug-treated MCTS or control
Results
1. Endpoint bright field and fluorescent images and results of MCTS for control and treated sample
- The bright field images were used to identify the spheroids in each well
- The Caspase 3/7 fluorescent intensities were measured from the images
- The plot below is showing the Caspase 3/7 fluorescent intensities, which indicated the apoptosis of each MCTS treated with the different drug compounds
- Only 2 drug compounds induced noticeable apoptosis on the U87MG MCTS, while other drugs had no effects
Conclusion
- Using the 384-well U-bottom ULA plates, we successfully captured images of U87MG MCTS and analyzed the data using the Celigo image cytometer
- The entire 384-well plate was imaged in ~8 min. The short scan time significantly increased the throughput during an experiment that has multiple plates
- After the drug treatments, the spheroid Caspase 3/7 fluorescent intensities were measured and automatically reported by the Celigo software
- No additional software was required for image analysis of MCTS fluorescent intensities