Sean Lauber:Using the Lewis Lung Carcinoma cell Mouse Model for Lung Cancer: Difference between revisions
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15. Spin tubes at 1,000 RPM for 3 min | 15. Spin tubes at 1,000 RPM for 3 min | ||
16. Discard supernatant, gently resuspend pellet by tapping | 16. Discard supernatant, gently resuspend pellet by tapping | ||
17. Combine resuspended pellets into a | 17. Combine resuspended pellets into a 50 ml falcon tube with 12 ml PBS | ||
18. Spin at 1,000 RPM for | 18. Spin at 1,000 RPM for 3 min, discard supernatant | ||
19. | 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 3,000 clumps to each animal (60,000 clumps/ml). I count a clump as being more than about 50 cells and should be easily recognized at 4X magnification. |
Revision as of 09:00, 18 September 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 3,000 clumps to each animal (60,000 clumps/ml). I count a clump as being more than about 50 cells and should be easily recognized at 4X magnification.