Mammary Adipocyte and Fibroblast Isolation

(adapted from Nranjan et al, 1995)

  1. Mince mouse mammary tissue.
  2. Partially digest tissue rotating for 1-2hr at 37oC in digestion cocktail.
    Digestion Cocktail
    1.5mg/ml Trypsin
    3mg/ml Collagenase A in MECL +10% FCS
    MECL Media:
    500mls DMEM/F12 (1:1)
    500µl (5µg/ml)  EGF
    500µl (10mg/ml) insulin

    For each gram of tissue, use 4mls of cocktail.

  3. Centrifuge digest for 30seconds at 3xg (so undigested material settle).
  4. Remove fat layer and plate in a small petri dish in DMEM + 10% FCS. ( The adipocytes will lose their fat droplet and adhere to the bottom of the plate.
    (The following day, remove the media and spin down cell. Add fresh media to cells plated the night before and plate the “new pellet” in DMEM +10% FCS.)
  5. Separate supernatant from settled material to a new tube.
  6. Spin at 190g for 10 min. Wash the pellet and seed in DMEM + 10% FCS.
  7. To get epithelium, take off the settled material (from 3) and redigest in fresh digestion cocktail for 30 min -1 hr at 37oC.
  8. Spin epithelial cells, wash 3-5X in PBS+2%FCS.
  9. Plate cells in MECL media.