The CellTiter-Glo® 3D Cell Viability Assay (Promega) was used to quantify spheroid viability on day 3. Samples were equilibrated to room temperature for at least 30 minutes prior to running the assay. A stock solution of 1:1 cell medium and CellTiter-Glo® was prepared, medium was removed from each sample well, and 400 μL of the stock solution was added to each sample.
Samples were protected from light and incubated for 1 hour on a shaker at room temperature. 100 μL triplicates were added to an opaque plate and luminescence was read immediately using a plate reader (BioTek Synergy HT Plate Reader and Gen5 software; BioTek Instruments, Inc.). A blank was prepared using stock solution. Relative luminescence for each sample was calculated by subtracting the average luminescence value from the blank wells from the average luminescence values of each sample.
Samples were protected from light and incubated for 1 hour on a shaker at room temperature. 100 μL triplicates were added to an opaque plate and luminescence was read immediately using a plate reader (BioTek Synergy HT Plate Reader and Gen5 software; BioTek Instruments, Inc.). A blank was prepared using stock solution. Relative luminescence for each sample was calculated by subtracting the average luminescence value from the blank wells from the average luminescence values of each sample.