Sean Lauber:Using the Lewis Lung Carcinoma cell Mouse Model for Lung Cancer: Difference between revisions
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7. Place at 37*C (with 5% CO2) for 72 h | 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 | 8. Recover the cells by pipetting and then ejecting the media onto the plate | ||
* repeat this twice and do it | * repeat this twice and do it 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) | 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 | 10. Bring the volume up to 10 ml with 10% FBS DMEM | ||
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14. Discard supernatant, gently resuspend pellet by tapping | 14. Discard supernatant, gently resuspend pellet by tapping | ||
15. Combine resuspended pellets into a single ependorrf | 15. Combine resuspended pellets into a single ependorrf | ||
16. Spin at 1,000 RPM for 1 min, discard supernatant, and bring up to final volume of approximately | 16. Spin at 1,000 RPM for 1 min, discard supernatant, and bring up to final volume of approximately 300 ul | ||
17. Gently tap the vial to resuspend the cells | 17. Gently tap the vial to resuspend the cells | ||
Ensure viability of 60-80% using trypan blue. | Ensure viability of 60-80% using trypan blue. 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 10X magnification. | ||
Revision as of 08:54, 13 September 2012
For 5 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 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 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 1,000 RPM for 3 min 14. Discard supernatant, gently resuspend pellet by tapping 15. Combine resuspended pellets into a single ependorrf 16. Spin at 1,000 RPM for 1 min, discard supernatant, and bring up to final volume of approximately 300 ul 17. Gently tap the vial to resuspend the cells
Ensure viability of 60-80% using trypan blue. 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 10X magnification.
