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Publisher Summary This chapter discusses the method for sequential enzyme treatment of mouse mammary gland. This method first weakens, without completely digesting, the structural constituents of the stroma by collagenase and hyaluronidase, and then in a second step, a general proteolytic enzyme like Pronase or trypsin brings forth the complete dissolution of the stroma and also of cell adhesion, resulting in a monocellular suspension. An important feature of the method, further, is that no means of mechanical disruption is applied during the enzyme treatments. Vigorous repeated pipetting used for the final disruption in many enzymatic methods is the main reason for low viability. The only mechanical step in the method is the initial dissection of the tissue. The cell yield by this method approximates the maximum attainable. If cultured on glass, the 2.5% Pronase-isolated cells show epithelium-like growth; in cultures of 1.25% Pronase-isolated cells, there is admixture of fibroblastlike cells. The method has also been used for the preparation of cell suspensions from mammary tumors. |
