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:

• 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