Difference between revisions of "Myotube preparation of C2C12 cells"
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==Procedure:== | ==Procedure:== | ||
| − | + | * open 3 of the conventional tubes (label one of this with 100 and the other two with 20) | |
| − | + | * 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 | |
| − | + | * take the same with the two 20µm filters and the two tubes (labeled with 20) | |
| − | + | * pipette in two of the 6 cm dishes 2 ml of HS-LS | |
| − | + | * label the other two with FT-A and FT-B (FT= flow through) | |
| − | + | * 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) | |
| − | + | * aspirate the PBS | |
| − | + | * pipette 2 ml TE to the cells and let this incubate till the myotubes are detached in large pieces | |
| − | + | * quickly stop the TE with 6 ml of HS-LS | |
| − | + | * pipette the suspension up and down till the myotubes are completely detached | |
| − | + | * pipette the cell suspension drop wise through the 100µm filter | |
| − | + | * wash the filter with 2 ml HS-LS | |
| − | + | * remove the filter from the tube | |
| − | + | * drip a small volume of HS-LS through the 20 µm filter | |
| − | + | * pipette the filtrate drop wise through the 20 µm filter | |
| − | + | * wash the filter with 2 ml of HS-LS | |
| − | + | * place the filter up side down into one of the both 6cm dishes with the 2 ml HS-LS | |
| − | + | * wash the filter with 2 ml HS-LS and then discard the filter | |
| − | + | * take the filtrate from the this 20 µm filter in the 6cm dish, which is label with FT-A (put this in the incubator) | |
| − | + | * drip a small volume of HS-LS through the second 20 µm filter | |
| − | + | * pipette the cell suspension from the 6cm dish (this one in which the filter were) drop wise through the second 20 µm filter | |
| − | + | * 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 | |
| − | + | * wash the filter with 2 ml HS-LS and then discard the filter | |
| − | + | * pipette the cell suspension into the 4th conventional tube | |
| − | + | * wash the dish with some HS-LS and pipette this also in the 4th tube | |
| − | + | * fill all up to 15 ml with HS-LS | |
| − | + | * remove the fibronectin from the 96 well plate | |
| − | + | * take the 5ml Multipipette and cut with the scissors the tip of this pipette | |
| − | + | * open the tip a little bit with a tweezers | |
| − | + | * mix the cells carefully (don’t produce air bubbles) with the Multipipette | |
| − | + | * pipette in each well 150 µl of the cell suspension | |
| − | + | * check the plate under the microscope | |
| − | + | * take the plate into the incubator | |
| − | + | * check the prep the next day under the inverted microscope | |
Revision as of 09:09, 3 September 2014
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:
- open 3 of the conventional tubes (label one of this with 100 and the other two with 20)
- 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
- take the same with the two 20µm filters and the two tubes (labeled with 20)
- pipette in two of the 6 cm dishes 2 ml of HS-LS
- label the other two with FT-A and FT-B (FT= flow through)
- 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)
- aspirate the PBS
- pipette 2 ml TE to the cells and let this incubate till the myotubes are detached in large pieces
- quickly stop the TE with 6 ml of HS-LS
- pipette the suspension up and down till the myotubes are completely detached
- pipette the cell suspension drop wise through the 100µm filter
- wash the filter with 2 ml HS-LS
- remove the filter from the tube
- drip a small volume of HS-LS through the 20 µm filter
- pipette the filtrate drop wise through the 20 µm filter
- wash the filter with 2 ml of HS-LS
- place the filter up side down into one of the both 6cm dishes with the 2 ml HS-LS
- wash the filter with 2 ml HS-LS and then discard the filter
- take the filtrate from the this 20 µm filter in the 6cm dish, which is label with FT-A (put this in the incubator)
- drip a small volume of HS-LS through the second 20 µm filter
- pipette the cell suspension from the 6cm dish (this one in which the filter were) drop wise through the second 20 µm filter
- 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
- wash the filter with 2 ml HS-LS and then discard the filter
- pipette the cell suspension into the 4th conventional tube
- wash the dish with some HS-LS and pipette this also in the 4th tube
- fill all up to 15 ml with HS-LS
- remove the fibronectin from the 96 well plate
- take the 5ml Multipipette and cut with the scissors the tip of this pipette
- open the tip a little bit with a tweezers
- mix the cells carefully (don’t produce air bubbles) with the Multipipette
- pipette in each well 150 µl of the cell suspension
- check the plate under the microscope
- take the plate into the incubator
- check the prep the next day under the inverted microscope