Colony formation assay

MCF-10A cells were plated at 3 × 10⁴ cells per well and MDA-MB-231 were plated at 5 × 10³ cells per well of a six well plate. The wells were first coated in 1.5 mL of 0.8% agarose diluted in normal culture media. Cells were plated in 1.5 mL of 0.4% agarose diluted in normal culture media and the agarose was allowed to set at room temperature for 1 hr. Samples were cultured for 4 weeks with 0.5 mL culture media added on top of the culture media with media exchanges every 2-3 days. After 4 weeks, samples were fixed in 10% formalin for 30 minutes and incubated in .005% crystal violet for 1 hr. Samples were rinsed in water until the washes were clear of stain. Images were acquired using a ChemiDoc XRS+ gel imager (BioRad) and associated software. Colonies were counted using ImageJ.