Cellular proliferation rate in different media and drug conditions was determined as previously described (Sullivan et al., 2015 (link)). Briefly, cell lines proliferating in log phase in RPMI medium were trypsinized, counted and plated into six well dishes (Corning Life Sciences) in 2 mL of RPMI medium and incubated overnight. Initial seeding density was 20,000 cells/well for A549 cells, or 50,000 cells for MCF7, AU565 and MDA-MB-468 cells. The next day, a six well plate of cells was trypsinized and counted to provide a number of cells at the start of the experiment. Cells were then washed twice with 2 mL of phosphate buffered saline (PBS), and 8 mL of the indicated media premixed with indicated compounds or vehicles was added. This large volume of media was chosen to prevent severe nutrient depletion, especially when adding adult bovine serum medium. Cells were then trypsinized and counted 4 days after adding the indicated medias. Proliferation rate was determined using the following formula: Proliferation rate in doublings/day = [Log2(Final Day 4 cell count/Initial Day 0 cell count)]/4 days. Cells were counted using a Cellometer Auto T4 Plus Cell Counter (Nexcelom Bioscience, Lawrence, MA).
Free full text: Click here
