Mammary Tumor Implantation

Materials

  • Collagenase: Roche #11088793001
  • DNase I (Roche 10104159001;100mg @ 2000U/mg): Stock at 25 U/ul
  • 70um Filter: BD Falcon #352350
  • Matrigel: BD #354230 GFR/LDEV-Free 10mls

Cell Preparation

  1. Harvest R2/3 tumors from several late-stage PyMT animals following whole body perfusion with 10-15 mls PBS/Heparin. Place tumors in ice-cold PBS until all are resected.
  2. Mince tumors in petri dish and transfer to 50 mL pyrex container with stir bar.
  3. Add Collagenase (2 mg/ml) and DNAse (2 ul/ml) and digest for 30-45 min at 32-35°C.
  4. Pass mixture through 70 mm filter (may need several).
  5. Spin and remove supernatant. Resuspend cells in 50 ml total DMEM/10%FCS.
  6. Pass through 70 mm filter once more. Centrifuge and resuspend in 50 ml DMEM/10%FCS.
  7. Count cells. Resuspend at 4x106 per mL in 90%FCS/10%DMSO and transfer to -80ºC for 1 day.
  8. Transfer cells to LN2 for long-term storage. 

Injection

  1. Thaw matrigel on ice O/N. Will need 50 ul per injection.
  2. Thaw cells and transfer to 10 ml DMEM/10% FCS. Spin and remove supernatant.
  3. Resuspend cells at 106 per 50 ul for each mouse to be injected.
  4. Add equal volume of matrigel. Keep cells on ice.
  5. Anesthetize mice for injection.
  6. Transfer cells to a 500ul insulin syringe.
  7. Spray down the injection site with 70% EtOH (usually R2/3 or R4/5 gland)
  8. Lift up skin/gland and insert needle bevel side up. 
  9. Slowly inject 100 ul volume, slowly remove needle, and pinch skin for 15 s to prevent leakage.
  10. Tumor should be visible by ~2 weeks post-injection.