Once week prior to fusion start Rhodamine-6-G treatment of muscle. At the same time plate 2x10^5 of donor cells (5 plates).
1. The day before the actual fusion prepare the following Ficoll gradient (containing cytochalasin D or B for enucleation)
Ficall Final conc (%) 50% Ficoll stock 4xDMEM dH2O Cyto D (100ug/ml)
25 12.5ml 6.4ml 6.0ml 100
20 10.0ml 6.4ml 8.5ml 100
17 8.5ml 6.4ml 10ml 100
15 7.5ml 6.4ml 11ml 100
12.5 6.25ml 6.4ml 12.25ml 100
Note: dissolve cyto D in DMSO first and then dilute with dH2O.
2. Layer the following amounts of the ficoll dilutions in the test tube: bottom of tube: 1.5ul of 25%; 1.5ul of 20%; 1.5ul of 17%; 0.5ul of 15%; 0.5ul of 12.5%.
3. Mark the gradient borders of the test tube and let the gradient equilibrate
4. Harvest 3-5 x 10^7 of 3243 cells (this is critical as greater than 5x 10^7 cells will clump and lead to poor enucleation). Replace old medium by spinning down cells and resuspending in 10 ml of resh DMEM / 10% FCS. Preheat ultracentrifuge at 37c.
5. Add Actinomycin D (100ug/ml stock) 100ul/10 ml of medium.
6. Incubate at 37c for 1 hour.
7. Pellete cells and resuspend in 3.0 ml of 12.5% ficoll with cyto D (as used in gradient preparation). Pipette well to prevent clumping and incubate for 1 hour.
8. Lyer this solution on top of ficoll layer.
9. Spin in ultracentrifuge at 28,000 rpm for 1 hour at 37c.
10. Remove tubes form centrifuge and wipe with alcohol.
11. Collect all ficoll slowly until just above sharp line. If no sharp line is observed collect no deeper than the 3rd line from the top.
12. Fill the tube with medium (without FBS) and pellet cytoplasts for 5 min at 1300 rpm.
13. Trypsinise the rhodamine treated cells to be used as nuclear background. Resuspend in DMEM/15% FBS and transfer into tube with cytoplast pellet. Mix.
14. Spin 1300 rpm for 5min. Remove supernatant completely.
15. Put some polyethylene glycol on top of pellet. Resuspend carefully but leave some chunks.
16. After exactly 1 min, dilute PEG with 10 ml of DMEM (no serum). Resuspend pellet carefully but no completely.
17. Spin for 4 min at 1300 rpm.
18. Wash 2x DMEM /10% FBS for 10min at 1300 rpm.
19. Resupend in 2 ml of final medium, but don't dissolve chunks completely.
20. Incubate for 1 hour at 37c, then resuspend.
21. Add 8ml of medium and palte. The selective media for muscle in DMEM/15%/UrHAT.