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

For 5 animals:

1. Thaw a single vial of 1,000,000 cells
2. Add to 9 ml of warmed 10% FBS DMEM
3. Spin at 10,000 RPM for 3 min
4. Discard supernatant
5. Resuspend to 10 ml
6. Plate this volume on a single 100 mm non-tissue culture treated petri dish 
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 VERY GENTLY to ensure you don't disturb the clumps
9. Split 1:3 (put 1/3 volume back onto original plate, and the other 1/3 onto two new plates)
10. Bring the volume up to 10 ml with 10% FBS DMEM
11. Place at 37*C (with 5% CO2) for 24 h
12. Collect cells as in step 8 into 50 ml falcon tubes
13. Use an additional 5-10 ml of 10% FBS DMEM to pick up the remaining cells
13. Spin tubes at 10,000 RPM for 3 min
14. Discard supernatant, gently resuspend pellet by tapping
15. Combine resuspended pellets into a single ependorrf
16. Spin at 10,000 RPM for 1 min, discard supernatant, and bring up to final volume of approximately 150-200 ul
17. Gently tap the vial to resuspend the cells

Ensure viability of 60-80% using trypan blue. Use 25 ul of cells (+25 ul of cytokine) to perform intubations.