Quantitative Colony Formation Assay

Cells were seeded in 6-well plates (500–100 cells/well). After 10 days, cells were fixed and stained using the sulforhodamine B (SRB) protocol, as described^{46 (link),50 (link)}. In brief, cells were fixed at 4 °C for 1 h using Trichloroacetic acid (TCA) (Sigma), washed then stained on a rocking platform for 30 min with 0.04% SRB in 1% acetic acid. Plates were imaged using Zeiss Observer Z1, ×5/0.5NA objective and Hamamatsu Flash V3 cMOS camera. Motorized stage was calibrated to tile-image the entirety of each well. Stitched images were analyzed in ImageJ/FIJI using customized macro script. Gaussian and median filters were used to blur the image before colonies were detected by intensity threshold. Touching colonies were separated using Watershed algorithm. The number of colonies and the area of each were analyzed. Quantification of the number of colonies per well and colony size was performed using ImageJ. Experiments were performed in triplicate.