Myotube preparation of C2C12 cells

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Revision as of 09:09, 3 September 2014 by Stephanh (talk | contribs)
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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