Epicult b basal medium

Single cell preparations of fetal mammary cells were obtained by pooling freshly dissected fetal mammary rudiments from euthanized embryos into dissociation media: Epicult-B Basal medium (Stem Cell Technologies) supplemented with 5% FBS, pen/strep, fungizone, hydrocortisone, collagenase and hyaluronidase. Rudiments were then dissociated to single cells by sequentially incubating them in dissociation medium for 1.5 hours at 37°C with gentle agitation, exposing them to ammonium chloride for 4 minutes on ice to remove erthyrocytes, and triturating them with dispase and DNase. Final suspensions were passed through a 40 um filter to remove aggregated cells, and stored in Hank’s Balanced Salt Solution with 2% FBS for flow cytometry. Single cell preparations of adult mammary cells were prepared by dissecting out and mincing the #4 mammary glands from 6–12 week old virgin female mice. Glands were then dissociated by agitating them for 3–6 hours at 37°C in the same dissociation media. Cells were further processed as with the fetal cells, except that trypsin and accutase (Life Technologies) were also utilized prior to dispase treatment to facilitate disaggregation. Final suspensions were passed through a 40 um filter to remove cell clusters, and stored in Hank’s Balanced Salt Solution with 2% FBS for flow cytometry.

Mammary tumors were dissected out of freshly euthanized mice, minced into small pieces, placed in dissociation media (Epicult-B Basal medium (Stem Cell Technologies) supplemented with 5% FBS, pen/strep, fungizone, hydrocortisone, collagenase and hyaluronidase), and agitated with shaking for 3 hours at 37°C. Next, erthyrocytes were removed with ammonium chloride exposure for 4 minutes on ice, followed by treatment and trituration with dispase and DNase. Final suspensions were passed through a 40 um filter to remove aggregated cells, and stored in Hank’s Balanced Salt Solution with 2% FBS for flow cytometry.