Collagenase solution preparation

A 0.025 % (w/v) solution of Collagenase type n, activity 242 U/mg, from Worthington, corresponding to an activity of 6 U/ml in Ham s F12 is prepared, filtered under sterile conditions, and supplemented with 10% FCS. 

Prepare all solutions using ultrapirre water and analytical grade reagents. Store all buffers perform ultracentrifugation and preparation of MVs at 4 °C. Treatment with collagenase is performed at 37 °C. 

Tools, tubes, 3.5 cm dish, PBS, Trypsin, collagenase, 37 °C water bath should be ready. 1 mg/ml trypsin-PBS solution [Sigma T-47999] (5 ml per a mouse x 3 runs). 2 mg/ml collagenase/PBS solution [Worthington type2, 4176] (1 ml per a mouse x2 runs), (both prepared and sterilized by 0.2 pM filter on the day). 

Normal animal tissues (e.g., skin, kidney, liver) or whole embryos are commonly used to establish primary cell cultures. To prepare tissue cells for a primary culture, the cell-cell and cell-matrix interactions must be broken. To do so, tissue fragments are treated with a combination of a protease (e.g., trypsin or the collagen-hydrolyzing enzyme collagenase or both) and a divalent cation chelator (e.g., EDTA) that depletes the medium of usable Ca or Mg. The released cells are then placed in dishes in a nutrient-rich, serum-supplemented medium, where they can adhere to the surface and one another. The same protease/chelator solution is used to remove adherent cells from a culture dish for biochemical studies or subculturing (transfer to another dish). 

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