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