Assay Protocol and Plate Setup
Monitor the spheroid growth and intensity of PI in U87MG MCTS for 10 days
- Seeded different cell numbers from 3,200 to 100 U87MG cells/well in a ULA U-bottom 96-well plate
- Added 2X PI staining solution and vehicle control in media to the appropriate wells
- Incubated the plate at 37 °C and 5% CO2
- Imaged and analyzed on Celigo for 10 days
- Analyzed the PI intensities and size of the spheres for 10 days
- After forming MCTS with PI, the plate was imaged in BF and Red illuminations and analyzed daily for 10 days. Each scan took ~ 2 minutes.
- The images were analyzed by using Tumorsphere 1 + Mask application
- The PI fluorescent intensities and sphere diameters were measured for the MCTS
Figure 1. Bright field and red fluorescent images of U87MG MCTS with PI (red) over 10 days
Figure 2. PI dead cell signal intensities and sphere diameters over 10 days.
Line graph showing the trends of PI intensity for different cell concentration for 10 days (a). Line graph showing that there is no toxic effect of PI on MCTS diameter (b).