- 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
- 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.
- Mince tumors in petri dish and transfer to 50 mL pyrex container with stir bar.
- Add Collagenase (2 mg/ml) and DNAse (2 ul/ml) and digest for 30-45 min at 32-35°C.
- Pass mixture through 70 mm filter (may need several).
- Spin and remove supernatant. Resuspend cells in 50 ml total DMEM/10%FCS.
- Pass through 70 mm filter once more. Centrifuge and resuspend in 50 ml DMEM/10%FCS.
- Count cells. Resuspend at 4x106 per mL in 90%FCS/10%DMSO and transfer to -80ºC for 1 day.
- Transfer cells to LN2 for long-term storage.
- Thaw matrigel on ice O/N. Will need 50 ul per injection.
- Thaw cells and transfer to 10 ml DMEM/10% FCS. Spin and remove supernatant.
- Resuspend cells at 106 per 50 ul for each mouse to be injected.
- Add equal volume of matrigel. Keep cells on ice.
- Anesthetize mice for injection.
- Transfer cells to 1 ml syringe. Attached 255/8 gauge needle.
- Spray down the injection site with 70% EtOH (usually R2/3 or R4/5 gland)
- Lift up skin/gland and insert needle bevel side up.
- Slowly inject 100 ul volume, slowly remove needle, and pinch skin for 15 s to prevent leakage.
- Tumor should be visible by ~2 weeks post-injection.