Name: Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation (Video) | JoVE Journal
Uploaded: 2009-10-06T16:30:00.000+00:00
Duration: 4 min 51 s
Description: Learn to induce lactogenic differentiation in HC11 mouse mammary epithelial cells and quantify mammosphere formation as a phenotypic marker. This protocol enables reliable in vitro study of mammary gland biology.

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5 minutes

Funds from Congressionally Directed Medical Research Fund grant (DAMD17-01-0264) to M.L. Cutler and United States Military Cancer Institute to M.L. Cutler supported the work.

<ol>
<li>The HC11 cells were grown to confluence for 6 days in RPMI 1640 medium supplemented with 10% fetal calf serum, 5μg/ml Insulin, 10 mM HEPES, 10ng/ml epidermal growth factor (EGF) to establish competence.</li>
<li>The cells were washed with PBS and maintained in growth media without EGF for 24 hours.</li>
<li>To induce lactogenic differentiation the cells were incubated in differentiation media, i.e. RPMI with dexamethasone (10-6 M), insulin (5 μg /ml) and prolactin (5 μg /ml) referred to as DIP.</li>
<li>At the stated times post-DIP addition, mammospheres were photographed and enumerated by phase contrast microscopy using an Olympus IX71 microscope with digital camera.</li>
</ol>
Representative Results:
Low power magnification of normal mammary epithelial cell monolayers and multiple mammospheres, as well as high power magnification of a single mammosphere are shown in Figure 1, respectively. <img src="/files/ftp_upload/1265/1265fig1.jpg" alt="Mammosphere formation, microscopy image; normal monolayer vs. multiple spheres, high magnification." data-altupdatedat="1787630408000"> Figure 1. Mammospheres. (a) Normal mammary epithelial cell monolayers should be displayed as a flat layer of confluent cells. (b) After 3-5 days of DIP stimulation multiple mammospheres appear.  Photograph taken 5 days post DIP addition. (c) Higher magnification of a single field containing mammospheres at 5 days post DIP addition.

<ol><li>Blatchford, D. R., Hendry, K. A., Turner, M. D., Burgoyne, R. D., Wilde, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vectorial+secretion+by+constitutive+and+regulated+secretory+pathways+in+mammary+epithelial+cells%5BTitle%5D)+AND+%22Epithelial+Cell+Biol%22%5BJournal%5D)">Vectorial secretion by constitutive and regulated secretory pathways in mammary epithelial cells.</a> Epithelial Cell Biol. 4, 8-16 (1995).</li>
<li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucocorticoid+induced+expression+of+connective+tissue+growth+factor+contributes+to+lactogenic+differentiation+of+mouse+mammary+epithelial+cells%5BTitle%5D)+AND+%22J+Cell+Physiol%22%5BJournal%5D)">Glucocorticoid induced expression of connective tissue growth factor contributes to lactogenic differentiation of mouse mammary epithelial cells.</a> J Cell Physiol. 214, 38-46 (2008).</li>
</ol>

In order for this technique to be successful and of the best quality, the epithelial cells must be maintained at confluence prior to being induced with lactogenic hormones. There may be situations where there are numerous mammospheres, which makes the quantification difficult. Thus, for quantitative purposes it is best to photograph the mammospheres at a time point when the number of mammospheres is able to be counted.
While there are various molecular markers used to determine the degree of ...

<table><tbody><tr><td>RPMI</td><td>Invitrogen</td><td>21870076</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>L-Glutamine</td><td>GIBCO, by Life Technologies</td><td>25030</td><td></td></tr><tr><td>PenStrep</td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>Hepes</td><td>Quality Biological, Inc.</td><td>118089060</td><td></td></tr><tr><td>EGF</td><td>Sigma-Aldrich</td><td>E9644</td><td></td></tr><tr><td>Insulin</td><td>GIBCO, by Life Technologies</td><td>12585-014</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D1756</td><td></td></tr><tr><td>Microscope</td><td>Olympus Corporation</td><td>IX71</td><td></td></tr><tr><td>Digital Camera</td><td>QImaging</td><td>Retiga 2000RV Fast 1394</td><td></td></tr></tbody></table>

mouse mammary epithelial cells form mammospheres during lactogenic differentiation

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

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.

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Learn to induce lactogenic differentiation in HC11 mouse mammary epithelial cells and quantify mammosphere formation as a phenotypic marker. This protocol enables reliable in vitro study of mammary gland biology.

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

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