Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation
Summary
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.
Abstract
Protocol
- 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.
- The cells were washed with PBS and maintained in growth media without EGF for 24 hours.
- 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.
- At the stated times post-DIP addition, mammospheres were photographed and enumerated by phase contrast microscopy using an Olympus IX71 microscope with digital camera.
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.
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.
Discussion
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 lactogenic differentiation of mammary epithelial cells, there are few morphological markers used for cells grown in 2D culture. The ability of epithelial cell monolayers to form miniature dome structures where milk proteins can accumulate provides a mode by which the morphological changes that occur during this developmental process can be monitored.
Acknowledgements
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.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| RPMI | Invitrogen | 21870076 | ||
| Fetal Bovine Serum | Atlanta Biologicals | S11550 | ||
| L-Glutamine | Gibco | 25030 | ||
| PenStrep | Gibco | 15140 | ||
| Hepes | Quality Biologicals | 118089060 | ||
| EGF | Sigma | E9644 | ||
| Insulin | Gibco | 12585-014 | ||
| Dexamethasone | Sigma | D1756 | ||
| Microscope | Olympus | IX71 | ||
| Digital Camera | QImaging | Retiga 2000RV Fast 1394 |
Referencias
- Blatchford, D. R., Hendry, K. A., Turner, M. D., Burgoyne, R. D., Wilde, C. J. Vectorial secretion by constitutive and regulated secretory pathways in mammary epithelial cells. Epithelial Cell Biol. 4, 8-16 (1995).
- Wang, W. Glucocorticoid induced expression of connective tissue growth factor contributes to lactogenic differentiation of mouse mammary epithelial cells. J Cell Physiol. 214, 38-46 (2008).
