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

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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 30,000 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.