View source for Sean Lauber:Using the Lewis Lung Carcinoma cell Mouse Model for Lung Cancer

For 15 animals:
 1. Thaw a single vial of 3,000,000 cells
 2. Add to 9 ml of warmed 10% FBS DMEM
 3. Spin at 1,000 RPM for 3 min
 4. Discard supernatant
 5. Resuspend to 9 ml
 6. Plate 3 ml onto a single 100 mm non-tissue culture treated petri dish (3 dishes for 9 ml)
 7. Bring to a final volume of 15 ml (per plate)
 7. Place at 37*C (with 5% CO2) for 72 h
 8. Recover the cells by pipetting and then ejecting the media onto the plate
 * repeat this twice and do it gently to ensure you don't disturb the clumps
 9. Remove 15 ml from each plate and place into a 50 ml falcon tube
 10. Take 5 ml of media and wash off remaining cells on the plates, adding it to falcon tube
 11. To split cells 1:3, add 5 ml of media to 9 petri dishes, then ad 5 ml of cells (from falcon tube)
 12. Place at 37*C (with 5% CO2) for 24 h
 13. Collect cells as in step 8 into 50 ml falcon tubes
 14. Use an additional 5-10 ml of 10% FBS DMEM to pick up the remaining cells
 15. Spin tubes at 1,000 RPM for 3 min
 16. Discard supernatant, gently resuspend pellet by tapping
 17. Combine resuspended pellets into a 50 ml falcon tube with 12 ml PBS
 18. Spin at 1,000 RPM for 3 min, discard supernatant
 19. Resuspend pellet by tapping, then add 500 ul PBS (final volume should now be 1 ml)


Aim for a concentration such that intubation of 50 ul delivers about 3000 clumps to each animal. I count a clump as being more than about 10 cells and should be easily recognized at 4X magnification. Cells must be resuspended (inverting tube) before each intubation (they settle out quickly). After 1 week the mice are sacrificed. Typically very few tumors are seen unless the mice are co-intubated with something pro-tumorigenic.