Sean Lauber:Using the Lewis Lung Carcinoma cell Mouse Model for Lung Cancer
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Aim for a concentration such that intubation of 50 ul delivers about
Aim for a concentration such that intubation of 50 ul delivers about ,000 clumps to each animal. I count a clump as being more than about cells and should be easily recognized at 4X magnification.
Revision as of 11:54, 7 December 2012
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.