Supplemental figures

Supplemental figure 1.- Linear correlation between Sytox intensity and cell density in 2D. A) Brightfield image of L929-2D culture. B) Sytox Green intensity for different concentrations of Sytox Green was plotted out against different cell densities. Linear correlation was measured as R² and the highest correlation was found for Sytox Green 3.3 µM. C) The increase of Sytox intensity for Sytox Green 3.3 µM is shown. Starting from 5000 cells, a high increase in intensity was found until 20 000 cells. D) Brightfield image of SKOV-2D culture. E) Sytox Blue intensity for different concentrations of Sytox Blue was plotted out against different cell densities. Linear correlation was measured as R² and the highest correlation was found for Sytox Blue 5 µM. F) The increase of Sytox intensity for Sytox Blue 5 µM is shown. Starting from 5000 cells, a high increase in intensity was found until 20 000 cells.

Supplemental figure 2.- Triton X-100 efficiently permeabilizes cells in 2D. L929-cells were stained with 3.3 µM of Sytox Green and permeabilised with Triton X-100 in different concentrations (0.05%, 0.10% and 0.25%). Sytox Green intensity was measured using the Tecan Spark microplate reader. Either top or bottom measurement was used, and intensity was measured at different timepoints.  A) Optimal gain setting. The highest linearity is found at gain 45. B) Comparison of top and bottom measurement. The most intense signal of Sytox Green with the lowest variation is found in top measurement. C) Optimal timing for measurement. No differences were observed at the different timepoints. D-F) Comparison of top and bottom measurement at different cell densities and with different Triton X-100 concentration. No differences were found between different Triton X-100 concentrations. RFU= relative fluorescent unit