Hi, I'm Bethany Morrison from the laboratory of Dr.Mary Lou Cutler in the Department of Pathology at the Uniform Services University of the Health Sciences. Today we'll be showing you a procedure for the development of mammos spheres from HC 11 mouse mammary epithelial cells. We use this procedure in our lab to study lacto differentiation.
So let's get started To start this protocol. HC 11 cells are grown to confluence for six days In RPMI 1640 medium, which is supplemented with 10%fetal calf serum five micrograms per milliliter insulin, 10 micromolar Heiss, and 10 nanograms per milliliter. Epidermal growth factor EGF to establish competence.
These cells have been used extensively by the laboratories of Nancy Hines and Bernard Groner to study the regulation of genic differentiation in the tissue culture hood. The cells are then washed with PBS and maintained in growth media without EGF for 24 hours. Following this incubation, the cells are incubated in differentiation media consisting of RPMI supplemented with dexamethasone insulin and prolactin in order to induce lacto differentiation after incubation.
In the differentiation media, an Olympus IX 71 microscope equipped with a digital camera is used to photograph and enumerate the mamo spheres. By phase contrast microscopy mamo spheres are photographed and enumerated multiple times through 120 hours post induction. This image shows a low power magnification of a normal memory.
Epithelial cell monolayer cells must be maintained at confluence for approximately three days prior to stimulation with genic hormones. Here is a look at new mammos spheres formed three days post induction at a low power magnification. They begin to appear as small dome like structures spaced randomly throughout the epithelial cells.
Again at low power. Here is a look at multiple mammo spheres that have developed five days post induction. The mamos spheres have continued to grow in both size and number here at high magnification, the bubble or dome like characteristic of the mamos sphere can be clearly seen.
We've just shown you how to induce memory epithelial cells, so they will form mammos spheres, which are used as markers of genic differentiation. When doing this procedure, it is important to remember to maintain the cells of confluence prior to induction because during this time, the cells produce matrix proteins with which they need to interact in order to be fully confident to respond to genic hormones. It's also important to remember to remove the EGF from the media prior to treatment because the EGF will inhibit the differentiation process.
So that's it. Thanks for watching and good luck with your experiments.
