Aside from determining the optimal numbers of DIPG cells to be seeded for viability assay, it is also important to validate that measurements using CellTiter Glo are in agreement with other methods of determining cell viability.
I chose to compare the EC50 values of M4K2009 on HSJD-DIPG-007 when measured using different methods. A simplified comparison of the different measurement methods is summarised in the following table.
1) 7 days of compound treatment gave the best assay window for EC50 estimated based on the confluency of the cells.
2) CellTiter Glo is superior over other methods of quantification in term of robustness and number of steps involved.
3) Propidium iodide staining of dead cells is not suitable for EC50 estimation because most dead cells likely became too degraded to be stained after 7 days of compound treatment.
4) EC50 estimation based on Calcein AM staining of metabolically active cells was robust but not as sensitive as CellTiter Glo or cell confluency.
5) EC50 estimation based on the amount of nuclei stained by Hoechst 33342 is relatively variable.
In a nut shell:
In future experiments, the EC50 of M4K compounds on the viability of DIPG patient-derived cell lines will be estimated based on CellTiter Glo signal and cell confluency.
For experimental details, please refer to my Zenodo page.