Myotube preparation of C2C12 cells

Materials

for 1 x 96 well plate:

• DMEM (3.5 ml) –purely
• Fibronectin (final diluted concentration should be 20 ng/µl))
• 1x 96 well plate
• 2% Horse Serum – Low Serum (HS-LS)
• 4 x 25 ml conventional tubes (white with top)
• 4 x 6 cm dishes
• 1x filter 100 µm
• 2x filter 20 µm
• 1 scalpel
• 2x tweezers
• 1x scissors
• Multipipetts and –tips (500µl, 5 ml)
• Pipettes
• PBS
• TE

Procedure

1. open 3 of the conventional tubes (label one of this with 100 and the other two with 20)
2. take the 100 µm filter on the tube (label with 100) and form a funnel with a hot tweezers and fix the filter with the hot scalpel (use the back of this) at three places
3. take the same with the two 20µm filters and the two tubes (labeled with 20)
4. pipette in two of the 6 cm dishes 2 ml of HS-LS
5. label the other two with FT-A and FT-B (FT= flow through)
6. carefully aspirate the old media from the differentiated cells and wash the cells with 5 ml PBS (pipette the PBS carefully without destroying the myotubes)
7. aspirate the PBS
8. pipette 2 ml TE to the cells and let this incubate till the myotubes are detached in large pieces
9. quickly stop the TE with 6 ml of HS-LS
10. pipette the suspension up and down till the myotubes are completely detached
11. pipette the cell suspension drop wise through the 100µm filter
12. wash the filter with 2 ml HS-LS
13. remove the filter from the tube
14. drip a small volume of HS-LS through the 20 µm filter
15. pipette the filtrate drop wise through the 20 µm filter
16. wash the filter with 2 ml of HS-LS
17. place the filter up side down into one of the both 6cm dishes with the 2 ml HS-LS
18. wash the filter with 2 ml HS-LS and then discard the filter
19. take the filtrate from the this 20 µm filter in the 6cm dish, which is label with FT-A (put this in the incubator)
20. drip a small volume of HS-LS through the second 20 µm filter
21. pipette the cell suspension from the 6cm dish (this one in which the filter were) drop wise through the second 20 µm filter
22. wash the filter with 2 ml HS-LS and place the filter up side down in the second 6 cm dish with the 2 ml HS-LS
23. wash the filter with 2 ml HS-LS and then discard the filter
24. pipette the cell suspension into the 4th conventional tube
25. wash the dish with some HS-LS and pipette this also in the 4th tube
26. fill all up to 15 ml with HS-LS
27. remove the fibronectin from the 96 well plate
28. take the 5ml Multipipette and cut with the scissors the tip of this pipette
29. open the tip a little bit with a tweezers
30. mix the cells carefully (don’t produce air bubbles) with the Multipipette
31. pipette in each well 150 µl of the cell suspension
32. check the plate under the microscope
33. take the plate into the incubator
34. check the prep the next day under the inverted microscope