Abstract
The mammosphere assay has become widely employed to quantify stem-like cells in a population. However, the problem is there is no standard protocol employed by the field. Cell seeding densities of 1,000 to 100,000 cells/mL have been reported. These high densities lead to cellular aggregation. To address this, we have individually tracked 1,127 single MCF-7 and 696 single T47D human breast tumor cells by eye over the course of 14 days. This tracking has given us detailed information for the commonly used endpoints of 5, 7, and 14 days that is unclouded by cellular aggregation. This includes mean sphere sizes, sphere-forming efficiencies, and a well-defined minimum size for both lines. Importantly, we have correlated early cell division with eventual sphere formation. At 24 hr post seeding, we can predict the total spheres on day 14 with 98% accuracy in both lines. This approach removes cell aggregation and potentially shortens a 5- to 14-day assay to a 24 hours.